Immunoconjugates and methods

ABSTRACT

Immunoconjugates of the Formula (I) include a linking group for linking an antibody targeting ligand (Ab) to a drug (D). Embodiments of such immunoconjugates are useful for delivering the drug to selected cells or tissues, e.g., for the treatment of cancer. 
       Ab-[S-L 1 -L 2 -L 3 -L 4 -L 5 -L 6 -L 7 -D] n   (I)

INCORPORATION BY REFERENCE TO PRIORITY APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.17/812,869, filed Jul. 15, 2022, which claims priority to U.S.Provisional Application Ser. No. 63/203,347, filed Jul. 19, 2021; andU.S. Provisional Application Ser. No. 63/267,471, filed Feb. 2, 2022.The disclosures of each of the aforementioned applications are herebyexpressly incorporated herein by reference in their entireties.

SEQUENCE STATEMENT

The instant application contains a Sequence Listing, which has beensubmitted electronically and is hereby incorporated by reference in itsentirety. The sequence listing, was created on Sep. 28, 2022, is named2023-01-26 Sequence Listing-ZENO102P1.xml and is approximately 49,293bytes in size.

BACKGROUND Field

The application relates to conjugates that include a linking group forlinking an antibody targeting ligand to a cell-killing moiety (such as adrug), methods of making such conjugates, and methods of using suchconjugates to deliver the cell-killing moiety to selected cells ortissues, e.g., for the treatment or inhibition of a cancer.

Description

A number of antibody-drug conjugates (ADC) have been developed formedical uses. See, e.g., Nejadmoghaddam, M. et al., “Antibody-DrugConjugates: Possibilities and Challenges”, Avicenna J Med Biotech 11(1),3-23 (2019). The antibody in the ADC functions as a targeting agent todeliver the drug to a selected cell or tissue such as a cancer cell ortumor. In the United States, the U.S. Food and Drug Administration (FDA)has approved several ADC formulations, including inotuzumab ozogamicin(tradename BESPONSA), gemtuzumab ozogamicin (tradename MYLOTARG),brentuximab vedotin (tradename ADCETRIS), and ado-trastuzumab emtansine(tradename KADCYLA).

U.S. Pat. No. 10,155,821 discloses ADCs in which an antitumor compoundis conjugated to an anti-HER2 antibody via a linker. See also U.S.Patent Publication Nos. 2020/0385486 and 2019/0077880. Trastuzumabderuxtecan is an example of an ADC in which an anti-HER2 antibody(trastuzumab) is attached via a cleavable maleimide tetrapeptide linkerto an antitumor compound (deruxtecan). The FDA has approved aformulation known as fam-trastuzumab deruxtecan-nxki (tradename ENHERTU)for the treatment of adult patients with unresectable or metastaticHER2-positive breast cancer who have received two or more prioranti-HER2-based regimens in the metastatic setting. FIG. 1 illustratesthe manner in which it is believed the linker connects the antibody(mAb) to the drug moiety.

The FDA approvals represent milestones in the ongoing development oftherapeutic ADCs. However, there remains a need for improved ADCs tohelp address the long-felt need for additional options to treat cancerand/or deliver therapeutic payloads to selected cells or tissues.

SUMMARY

Some embodiments provide an immunoconjugate of Formula (I) thatcomprises an antibody or antigen-binding fragment (Ab), and drug moiety(D) and a linker connecting Ab to D. In an embodiment, theimmunoconjugate of Formula (I) comprises a drug moiety of the Formula(II).

An embodiment provides an immunoconjugate having Formula (I),

Ab-[S-L¹-L²-L³-L⁴-L⁵-L⁶-L⁷-D]_(n)   (I)

wherein:

-   -   Ab is an antibody or an antigen-binding fragment;    -   L¹ is

-   -   L² is absent,

-   -   Z¹ and Z² are each individually hydrogen, halogen, NO₂,        —O—(C₁-C₆ alkyl), or C₁-C₆ alkyl;    -   L³ is —(CH₂)n¹-C(═O)— or —(CH₂CH₂O)n¹-(CH₂)n¹C(═O)—;    -   n¹ are independently integers of 0 to 12;    -   L⁴ is a tetrapeptide residue;    -   L⁵ is absent or —[NH(CH₂)n²]n³-;    -   n² is an integer of 0 to 6;    -   n³ is an integer of 0 to 2;    -   L⁶ is absent or

-   -   L⁷ is absent,

-   -   D is a drug moiety; and    -   n is an integer from 1 to 10.

In an embodiment, D in Formula (I) is a drug moiety of Formula (II)having the structure:

-   -   wherein:

-   -   R¹ and R² are each individually selected from the group        consisting of hydrogen, halogen, —CN, —OR⁵, —NR⁵R⁶, a        substituted or an unsubstituted C₁-C₆ alkyl, a substituted or an        unsubstituted C₁-C₆ haloalkyl, a substituted or an unsubstituted        —O—(C₁-C₆ alkyl), a substituted or an unsubstituted —O—(C₁-C₆        haloalkyl), —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃, or a substituted or        an unsubstituted —O—(CR⁵R⁶)_(m)—O— such that R¹ and R² taken        together form a ring;    -   R³ is hydrogen or a substituted or an unsubstituted C₁-C₆ alkyl,        a substituted or an unsubstituted C₁-C₆ haloalkyl, or        —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃;    -   R⁴ is hydrogen, a substituted or an unsubstituted —(C₁-C₆        alkyl)-X², a substituted or an unsubstituted —(C₁-C₆        haloalkyl)-X², a substituted or an unsubstituted —(C₁-C₆        alkenyl)-X², a substituted or an unsubstituted —(C₁-C₆        haloalkenyl)-X², a substituted or an unsubstituted —(C₁-C₆        alkynyl)-X², or a substituted or an unsubstituted —(C₁-C₆        haloalkynyl)-X²;    -   X¹ is —O—, —S(O_(n6))—, —NH—, —O—(C═O)—, —NH—(C═O)—,        —NH—(C═O)—O—, —NH—(C═O)—NH—, or —NH—S(O_(n6))—;    -   X² is —OR⁹, —SR⁹, or —NHR⁹;    -   R⁵ and R⁶ are each individually hydrogen, halogen, a substituted        or an unsubstituted C₁-C₆ alkyl, a substituted or an        unsubstituted C₁-C₆ haloalkyl, or —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃;    -   m is 1 or 2;    -   n⁴ and n⁵ are each individually 0, 1 or 2, with the proviso that        n⁴ and n⁵ are not both 0;    -   n⁶ is 0, 1 or 2;    -   each Y is individually H or halogen;    -   each p is individually 1, 2, 3, 4, 5, or 6;    -   each q is individually 0, 1, 2, 3, 4, 5, or 6;    -   each t is individually 1, 2, 3, 4, 5, or 6;    -   R⁷ is H, —COR⁸, —CO₂R⁸, —(CO)—NHR⁸, L⁴, L⁵, L⁶, or L⁷;    -   R⁸ is a substituted or an unsubstituted C₁-C₆ alkyl-X³, a        substituted or an unsubstituted C₁-C₆ haloalkyl-X³, or        —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₂—X³;    -   R⁹ is H, —COR⁸, —CO₂R⁸, —(CO)—NHR⁸, L⁴, L⁵, L⁶, or L⁷, with the        proviso that exactly one of R⁷ and R⁹ is L⁴, L⁵, L⁶, or L⁷; and    -   each X³ is individually —H, —OH, —SH, or —NH₂.

An embodiment provides a compound of Formula (IV), or a pharmaceuticallyacceptable salt thereof, having the structure:

-   -   wherein:    -   R¹ and R² are each individually selected from the group        consisting of hydrogen, halogen, —CN, —OR⁵, —NR⁵R⁶, a        substituted or an unsubstituted C₁-C₆ alkyl, a substituted or an        unsubstituted C₁-C₆ haloalkyl, a substituted or an unsubstituted        —O—(C₁-C₆ alkyl), a substituted or an unsubstituted —O—(C₁-C₆        haloalkyl), —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃, or a substituted or        an unsubstituted —O—(CR⁵R⁶)_(m)—O— such that R¹ and R² taken        together form a ring;    -   R³ is hydrogen or a substituted or an unsubstituted C₁-C₆ alkyl,        a substituted or an unsubstituted C₁-C₆ haloalkyl, or        —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃;    -   R⁴ is hydrogen, a substituted or an unsubstituted —(C₁-C₆        alkyl)-X², a substituted or an unsubstituted —(C₁-C₆        haloalkyl)-X², a substituted or an unsubstituted —(C₁-C₆        alkenyl)-X², a substituted or an unsubstituted —(C₁-C₆        haloalkenyl)-X², a substituted or an unsubstituted —(C₁-C₆        alkynyl)-X², or a substituted or an unsubstituted —(C₁-C₆        haloalkynyl)-X²;    -   X¹ is —O—, —S(O_(n6))—, —NH—, —O—(C═O)—, —NH—(C═O)—,        —NH—(C═O)—O—, —NH—(C═O)—NH—, or —NH—S(O_(n6))—;    -   X² is —OH, —SH, or —NR⁵R⁶;    -   R⁵ and R⁶ are each individually hydrogen, halogen, a substituted        or an unsubstituted C₁-C₆ alkyl, a substituted or an        unsubstituted C₁-C₆ haloalkyl, or —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃;    -   R⁷ is H, —COR⁸, —CO₂R⁸, or —(CO)—NHR⁸;    -   R⁸ is a substituted or an unsubstituted C₁-C₆ alkyl, a        substituted or an unsubstituted C₁-C₆ haloalkyl, or        —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃;    -   m is 1 or 2;    -   n⁴ and n⁵ are each individually 0, 1 or 2, with the proviso that        n⁴ and n⁵ are not both 0;    -   n⁶ is 0, 1 or 2; and    -   each Y is individually H or halogen;    -   each p is individually 1, 2, 3, 4, 5, or 6;    -   each q is individually 0, 1, 2, 3, 4, 5, or 6; and    -   each t is individually 1, 2, 3, 4, 5, or 6;    -   with the proviso that Formula (IV) does not represent deruxtecan        or exatecan.

An embodiment provides a pharmaceutical composition comprising animmunoconjugate as described herein, a drug compound as describedherein, or a pharmaceutically active salt thereof, and apharmaceutically acceptable carrier, diluent, excipient or combinationthereof.

An embodiment provides a method for treating a cancer or a tumorcomprising administering an effective amount of an immunoconjugate asdescribed herein, a drug compound as described herein, or apharmaceutically active salt thereof, or a pharmaceutical composition asdescribed herein, to a subject having the cancer or the tumor.

An embodiment provides a use of an effective amount of animmunoconjugate as described herein, a drug compound as describedherein, or a pharmaceutically active salt thereof, or a pharmaceuticalcomposition as described herein, in the manufacture of a medicament fortreating a cancer or a tumor.

Some embodiments provide a conjugate of Formula (III) that comprises afunctional group Ml, a drug moiety (D) and a linker connecting Mi to D.In an embodiment, the conjugate of Formula (III) comprises a drug moietyof the Formula (II).

An embodiment provides a conjugate having Formula (III),

Mi-L²-L³-L⁴-L⁵-L⁶-L⁷-D   (III)

-   -   wherein:    -   Mi is

-   -   L² is absent,

-   -   Z¹ and Z² are each individually hydrogen, halogen, NO₂,        —O—(C₁-C₆ alkyl), or C₁-C₆ alkyl;    -   L³ is —(CH₂)n¹-C(═O)— or —(CH₂CH₂O)n¹-(CH₂)n¹C(═O)—;    -   n¹ are independently integers of 0 to 12;    -   L⁴ is a tetrapeptide residue;    -   L⁵ is absent or —[NH(CH₂)n²]n³-;    -   n² is an integer of 0 to 6;    -   n³ is an integer of 0 to 2;    -   L⁶ is absent or

-   -   L⁷ is absent,

and

-   -   D is a drug moiety.

An embodiment provides a process of producing an immunoconjugate,comprising: reacting an effective amount of a thiol-functionalizedantibody or antigen-binding fragment with a conjugate as describedherein under reaction conditions effective to form an immunoconjugate asdescribed herein.

Preferred Alternatives Include:

-   -   1. An antibody or antigen-binding fragment thereof, comprising:        -   a) a heavy chain comprising:    -   VHCDR 1 comprising an amino acid sequence having at least 95%        sequence identity to the amino acid sequence of SEQ ID NO:1;    -   VHCDR 2 comprising an amino acid sequence having at least 95%        sequence identity to the amino acid sequence of SEQ ID NO:2; and    -   VHCDR 3 comprising an amino acid sequence having at least 95%        sequence identity to the amino acid sequence of SEQ ID NO:3; and    -   b) a light chain comprising:    -   VLCDR 1 comprising an amino acid sequence having at least 95%        sequence identity to the amino acid sequence of SEQ ID NO:8;    -   VLCDR 2 comprising an amino acid sequence having at least 95%        sequence identity to the amino acid sequence of AAS; and    -   VLCDR 3 comprising an amino acid sequence having at least 95%        sequence identity to the amino acid sequence of SEQ ID NO:10;    -   wherein the antibody or antigen-binding fragment thereof        specifically binds to the extracellular domain of human receptor        tyrosine kinase like orphan receptor 1 (ROR1).    -   2. The antibody or antigen-binding fragment of alternative 1,        comprising an amino acid sequence having at least 95% sequence        identity to the amino acid sequence of any one of SEQ ID NOs: 5,        7, 12, or 14.    -   3. One or more nucleic acids encoding the antibody or        antigen-binding fragment thereof of any one of alternatives 1 or        2, such as an antibody or antigen-binding fragment thereof        encoded by one or more nucleic acids comprising a sequence        having at least 95% sequence identity to the nucleic acid        sequence set forth in any one of SEQ ID NOs: 4, 6, 11, or 13.    -   4. A host cell comprising the one or more nucleic acids of        alternative 3.    -   5. The immunoconjugate of any one of the previous embodiments,        wherein Ab is the antibody or antigen-binding fragment of claim        alternatives 1 or 2.    -   6. An antibody or antigen-binding fragment thereof, comprising:    -   a) a heavy chain comprising:    -   VHCDR 1 comprising an amino acid sequence having at least 95%        sequence identity to the amino acid sequence of SEQ ID NO:15;    -   VHCDR 2 comprising an amino acid sequence having at least 95%        sequence identity to the amino acid sequence of SEQ ID NO:16;        and    -   VHCDR 3 comprising an amino acid sequence having at least 95%        sequence identity to the amino acid sequence of SEQ ID NO:17;        and    -   b) a light chain comprising:    -   VLCDR 1 comprising an amino acid sequence having at least 95%        sequence identity to the amino acid sequence of SEQ ID NO:22;    -   VLCDR 2 comprising an amino acid sequence having at least 95%        sequence identity to the amino acid sequence of DAY; and    -   VLCDR 3 comprising an amino acid sequence having at least 95%        sequence identity to the amino acid sequence of SEQ ID NO:24;    -   wherein the antibody or antigen-binding fragment thereof        specifically binds to the extracellular domain of human receptor        tyrosine kinase like orphan receptor 1 (ROR1).    -   7. The antibody or antigen-binding fragment of alternative 6,        comprising an amino acid sequence having at least 95% sequence        identity to the amino acid sequence of any one of SEQ ID NOs:        19, 21, 26 or 28.    -   8. One or more nucleic acids encoding the antibody or        antigen-binding fragment thereof of any one of alternatives 6 or        7, such as an antibody or antigen-binding fragment thereof        encoded by one or more nucleic acids comprising a sequence        having at least 95% sequence identity to the nucleic acid        sequence set forth in any one of SEQ ID NOs: 18, 20, 25, or 27.    -   9. A host cell comprising the one or more nucleic acids of        alternative 8.    -   10. The immunoconjugate of any one of previous embodiments,        wherein Ab is the antibody or antigen-binding fragment of        alternatives 6 or 7.    -   11. An antibody or antigen-binding fragment thereof, comprising:    -   a) a heavy chain comprising:    -   VHCDR 1 comprising an amino acid sequence having at least 95%        sequence identity to the amino acid sequence of SEQ ID NO:29;    -   VHCDR 2 comprising an amino acid sequence having at least 95%        sequence identity to the amino acid sequence of SEQ ID NO:30;        and    -   VHCDR 3 comprising an amino acid sequence having at least 95%        sequence identity to the amino acid sequence of SEQ ID NO:31;        and    -   b) a light chain comprising:    -   VLCDR 1 comprising an amino acid sequence having at least 95%        sequence identity to the amino acid sequence of SEQ ID NO:36;    -   VLCDR 2 comprising an amino acid sequence having at least 95%        sequence identity to the amino acid sequence of DAS; and    -   VLCDR 3 comprising an amino acid sequence having at least 95%        sequence identity to the amino acid sequence of SEQ ID NO:38;    -   wherein the antibody or antigen-binding fragment specifically        binds to the extracellular domain of human receptor tyrosine        kinase like orphan receptor 1 (ROR1).    -   12. The antibody or antigen-binding fragment of alternative 11,        comprising an amino acid sequence having at least 95% sequence        identity to the amino acid sequence of any one of SEQ ID NOs:        33, 35, 40 or 42.    -   13. One or more nucleic acids encoding the antibody or        antigen-binding fragment thereof of any one of alternatives 11        or 12, such as an antibody or antigen-binding fragment thereof        encoded by one or more nucleic acids comprising a sequence        having at least 95% sequence identity to the nucleic acid        sequence set forth in any one of SEQ ID NOs: 32, 34, 39, or 41.    -   14, A host cell comprising the one or more nucleic acids of        alternative 13.    -   15. The immunoconjugate of any one of the previous embodiments,        wherein Ab is the antibody or antigen-binding fragment of        alternatives 11 or 12.    -   16. An antibody or binding fragment thereof where the heavy        chain is encoded by the nucleic acid sequence set forth in SEQ        ID NO: 6 and the light chain is encoded by the nucleic acid        sequence set forth in SEQ ID NO: 13 or a composition comprising        said antibody or binding fragment thereof.    -   17. The antibody or binding fragment thereof of alternative 16        or a composition comprising said antibody or binding fragment        thereof, wherein the heavy chain of the antibody comprises the        polypeptide sequence of SEQ ID NO:7 and the light chain        comprises the polypeptide sequence of SEQ ID NO: 14.    -   18. The antibody or binding fragment thereof of any one of        alternatives 16 or 17 or a composition comprising said antibody        or binding fragment thereof, wherein said antibody or binding        fragment thereof is conjugated to a molecule.    -   19. The antibody or binding fragment thereof of alternative 18        or a composition comprising said antibody or binding fragment        thereof, wherein the molecule is a drug, toxin, or cytokine.    -   20. The immunoconjugate of anyone of claims 1-25, wherein said        antibody or binding fragment thereof is the antibody or binding        fragment thereof of any one of alternatives 16 or 17.    -   21. A method of using the antibody or binding fragment thereof        of any one of alternatives 16-19 or a composition comprising        said antibody or binding fragment thereof, such as the        immunoconjugate of alternative 20, for inhibiting or treating a        disease such as a cancer comprising administering the antibody        or binding fragment thereof of any one of alternatives 16-19 or        said composition to a subject in need thereof, optionally        selecting a subject to receive a therapy for said disease such        as cancer and/or optionally determining the inhibition of said        disease such as cancer after administration of said antibody or        binding fragment thereof.    -   22 The antibody or binding fragment thereof of any one of        alternatives 16-19 or a composition comprising said antibody or        binding fragment thereof, such as the immunoconjugate of        alternative 20, for use as a medicament, such as for the purpose        of inhibiting or treating a disease such as a cancer.    -   23. An antibody or binding fragment thereof where the heavy        chain is encoded by the nucleic acid sequence set forth in SEQ        ID NO: 20 and the light chain is encoded by the nucleic acid        sequence set forth in SEQ ID NO: 27 or a composition comprising        said antibody or binding fragment thereof.    -   24. The antibody or binding fragment thereof of alternative 23        or a composition comprising said antibody or binding fragment        thereof, wherein the heavy chain of the antibody comprises the        polypeptide sequence of SEQ ID NO:21 and the light chain        comprises the polypeptide sequence of SEQ ID NO: 28.    -   25. The antibody or binding fragment thereof of any one of        alternatives 23 or 24 or a composition comprising said antibody        or binding fragment thereof, wherein said antibody or binding        fragment thereof is conjugated to a molecule.    -   26. The antibody or binding fragment thereof of alternative 25        or a composition comprising said antibody or binding fragment        thereof, wherein the molecule is a drug, toxin, or cytokine.    -   27. The immunoconjugate of anyone of claims 1-25, wherein said        antibody or binding fragment thereof is the antibody or binding        fragment thereof of any one of alternatives 23 or 24.    -   28. A method of using the antibody or binding fragment thereof        of any one of alternatives 23-26 or a composition comprising        said antibody or binding fragment thereof, such as the        immunoconjugate of alternative 27, for inhibiting or treating a        disease such as a cancer comprising administering the antibody        or binding fragment thereof of any one of claims alternatives        23-26 or said composition to a subject in need thereof,        optionally selecting a subject to receive a therapy for said        disease such as cancer and/or optionally determining the        inhibition of said disease such as cancer after administration        of said antibody or binding fragment thereof.    -   29. The antibody or binding fragment thereof of any one of        alternatives 23-26 or a composition comprising said antibody or        binding fragment thereof, such as the immunoconjugate of        alternative 27, for use as a medicament, such as for the purpose        of inhibiting or treating a disease such as a cancer.    -   30. An antibody or binding fragment thereof where the heavy        chain is encoded by the nucleic acid sequence set forth in SEQ        ID NO: 34 and the light chain is encoded by the nucleic acid        sequence set forth in SEQ ID NO: 41 or a composition comprising        said antibody or binding fragment thereof.    -   31. The antibody or binding fragment thereof of alternative 30        or a composition comprising said antibody or binding fragment        thereof, wherein the heavy chain of the antibody comprises the        polypeptide sequence of SEQ ID NO:35 and the light chain        comprises the polypeptide sequence of SEQ ID NO: 42.    -   32. The antibody or binding fragment thereof of any one of        alternatives 30 or 31 or a composition comprising said antibody        or binding fragment thereof, wherein said antibody or binding        fragment thereof is conjugated to a molecule.    -   33. The antibody or binding fragment thereof of alternative 32        or a composition comprising said antibody or binding fragment        thereof, wherein the molecule is a drug, toxin, or cytokine.    -   34. The immunoconjugate of anyone of claims 1-25, wherein said        antibody or binding fragment thereof is the antibody or binding        fragment thereof of any one of alternatives 30 or 31.    -   35. A method of using the antibody or binding fragment thereof        of any one of alternatives 30-33 or a composition comprising        said antibody or binding fragment thereof, such as the        immunoconjugate of alternative 34, for inhibiting or treating a        disease such as a cancer comprising administering the antibody        or binding fragment thereof of any one of alternatives 30-33 or        said composition to a subject in need thereof, optionally        selecting a subject to receive a therapy for said disease such        as cancer and/or optionally determining the inhibition of said        disease such as cancer after administration of said antibody or        binding fragment thereof.    -   36. The antibody or binding fragment thereof of any one of        alternative 30-33 or a composition comprising said antibody or        binding fragment thereof, such as the immunoconjugate of        alternative 34, for use as a medicament, such as for the purpose        of inhibiting or treating a disease such as a cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a trastuzumab deruxtecan antibody-drug conjugate.

FIG. 2 illustrates a reaction scheme for making a compound of theFormula (IV). Under acidic or basic hydrolysis conditions, IV-2 isformed from IV-1. The Friedlander reaction of IV-2 and IV-3 affordsIV-4. Removal of the protecting group (Pg) in IV-4 affords IV-5. IV-5undergoes alkylation, esterification, or amidation to afford IV.

FIG. 3 illustrates a reaction scheme for making a compound of theFormula (IV-1a). The Heck reaction between IV-6 and IV-7 affords IV-8.IV-8 undergoes hydrogenation to afford IV-9, that is hydrolyzed toafford IV-10. Intramolecular Friedel-Crafts reaction of IV-10 affordsIV-11a. IV-11a undergoes alpha alkylation to afford IV-12 that istreated either with alpha-hydroxylation, or alpha-amination conditionsto afford IV-1a (when n⁵=0).

FIG. 4 illustrates a reaction scheme for making a compound of theFormula (IV-1b). Dehydrogenation of IV-11b affords IV-13. IV-13undergoes Michael reaction followed by dehydrogenation to afford IV-14.IV-14 undergoes Michael reaction using oxygen or nitrogen containingnucleophile to afford IV-1b (when n⁵=1).

FIG. 5 illustrates a reaction scheme for making a conjugate of theFormula (III) that comprises attaching a linking moiety to a compound ofthe Formula (IV). The reaction of IIIA-1 and IIIA-2 under heatingconditions affords IIIA-3. IIIA-3 reacts with N-hydroxysuccinimide toafford IIIA-4. The reaction of IIIA-4 and IIIA-5 affords IIIA-6. IIIA-6reacts with IIIA-7 under amide coupling conditions affording IIIA-8.

FIG. 6 illustrates a reaction scheme for making IIIA-13. The reaction ofIIIA-9 and N-hydroxysuccinimide (IIIA-10) affords IIIA-11, which reactswith IIIA-12 to afford IIIA-13.

FIG. 7 illustrates a reaction scheme for making IIIA-5. The reaction ofIIIA-14 with Pb(OAc)₄ affords IIIA-15. Treatment of IIIA-15 and IIIA-16in the presence of NaOH affords IIIA-17. IIIA-17 is treated with DBU toafford IIIA-18, which couples with IIIA-19 to afford IIIA-20.Hydrogenation of IIIA-20 affords IIIA-5.

FIG. 8 illustrates a reaction scheme for making IIIB-4. The reaction ofIIIA-4 and IIIB-1 in the presence of a base affords IIIB-2, which reactswith 4-amino benzyl alcohol and EEDQ to produce IIIB-3. The treatment ofIIIB-3 with 4-nitro phenyl chloroformate gives IIIB-4.

FIG. 9 illustrates a reaction scheme for making IIIB-13. The reaction ofIIIB-5 with DHP produces IIIB-6. IIIB-6 reacts with oxalyl chloride andcatalytic DMF to produce IIIB-7. The reaction of IIIB-7 and IIIA-7 givesIIIB-8, which reacts with 4-nitro phenyl chloroformate to afford IIIB-9.The reaction IIIB-9 and IIIB-10 in the presence of a base affordsIIIB-11. IIIB-11 is treated with TFA to afford IIIB-12. The combinationof IIIB-12 and IIIB-4 in the presence of a base affords IIIB-13.

FIG. 10 illustrates a reaction scheme for making IIIC-7. The reaction ofIIIC-1 with Pb(OAc)₄ produces IIIC-2, which reacts with IIIC-3 in thepresence of ZnCl₂ to afford IIIC-4. Treating IIIC-4 with DBU affordsIIIC-5. The amide coupling of IIIC-5 and IIIA-3 affords IIIC-6, whichreacts with HF-pyridine to afford IIIC-7.

FIG. 11 illustrates a reaction scheme for making an immunoconjugate ofthe Formula (I) that comprises attaching Ab to a conjugate of theFormula (III). The Michael reaction of the thiol group of cysteines fromthe antibody with the maleimide in Formula III produces Formula I.

FIG. 12 illustrates a reaction scheme for making compound 1-14.

FIG. 13 illustrates a reaction scheme for making compound 2-30.

FIG. 14 illustrates a reaction scheme for making compounds 3-34 and3-35.

FIG. 15 illustrates a reaction scheme for making compounds 4-36 and4-37.

FIG. 16 illustrates a reaction scheme for making compounds 5-47 and5-48.

FIG. 17 illustrates a reaction scheme for making compounds 3-34 and3-35.

FIG. 18 illustrates a reaction scheme for making compounds 7-59 and7-60.

FIG. 19 illustrates a reaction scheme for making compounds 8-71 and8-72.

FIG. 20 illustrates a reaction scheme for making compound 9-74.

FIG. 21 illustrates a reaction scheme for making compound 11-80.

FIG. 22 illustrates a reaction scheme for making compounds 13-84 and13-85.

FIG. 23 illustrates a reaction scheme for making compounds 14-91 and14-92.

FIG. 24 illustrates a reaction scheme for making compound 15-94.

FIG. 25 illustrates a reaction scheme for making compound 18-112.

FIG. 26 illustrates a reaction scheme for making compound 21-120.

FIG. 27 illustrates a measurement of cell binding saturation data forthe anti-ROR-1 antibodies generated by the methods described herein. AROR-1 positive cell line JeKo-1 was incubated in a titration series withthe anti-ROR-1 antibodies ATX-P-875, ATX-P-885, and ATX-P-890 incomparison to the positive control antibody UC961. Cells were washed,stained with secondary antibody and cell binding saturation was detectedby flow cytometry and reported as mean fluorescent intensity (MFI).

FIG. 28 illustrates ROR-1 receptor internalization data for theanti-ROR-1 antibodies ATX-875, ATX-P-885, ATX-P-890. ROR-1 positive celllines JeKo-1 and MDA-MB-468 were incubated with the anti-ROR-1antibodies ATX-P-875, ATX-P-885, and ATX-P-890 and positive controlantibody UC961 at super saturating conditions so as to bind allavailable ROR-1 receptors. Cells were washed and incubated at 4different timepoints (30 min, 1 hour, 2 hours and 4 hours) at 37° C.before internalization was halted by placing the cells in ice. Receptorinternalization was determined by flow cytometry and reported as percentreceptor internalization relative to zero hours.

FIG. 29A-29D illustrates cellular binning data for the anti-ROR-1antibodies ATX-P-875, ATX-P-885, and ATX-P-890. A cellular binning assaywas performed to assess if ATX-P-875, ATX-P-885, and ATX-P-890 bound thesame epitopes on the ROR-1 receptor as control antibodies UC961 and 4A5.(29A) depicts a staining profile for antibodies that bind the sameepitope. (29B) depicts the staining profile for antibodies that binddifferent epitopes. ATX-P-875, ATX-P-885, and ATX-P-890 were separatelyincubated with ROR-1_+ MDA-MB-468 at various amounts. Next, theanti-ROR-1 antibodies were fluorescently labeled with a secondaryantibody. Finally, MDA-MB-468 cells coated with the anti-ROR-1antibodies were incubated with a saturating dose of a fluorescentlylabeled UC961 (29C) or 4A5 (29D) and analyzed by flow cytometry and theATX-P-875, ATX-P-885, and ATX-P-890 antibody signal were compared withthe UC961 or 4A5 signal.

FIG. 30 illustrates AC-SINS data for the anti-ROR-1 antibodiesATX-P-875, ATX-P-885, and ATX-P-890. Antibody developability wasassessed by performing an AC-SINS assay and evaluating the potential forself-interaction. Rituximab and Infliximab were used as controls todemonstrate a low and high shift, respectively. Assay results forATX-P-875, ATX-P-885, and ATX-P-890 fell within the range determined bythe control antibodies.

FIG. 31 illustrates biochemical binning data by SPR for the anti-ROR1antibodies ATX-P-875, ATX-P-885, ATX-P-890 as compared against controlanti-ROR-1 antibodies UC961 (ATX-P-453) and 4a5.

FIG. 32 illustrates linker and payload combinations that were conjugatedto the 3 unique antibodies (mAb A=ATX-P-875, mAb B=ATX-P-885, mAbC=ATX-P-890). The antibodies, mAb A (ATX-P-875), mAb B (ATX-P-885), andmAb C (ATX-P-890) were conjugated to 6 separate novel linker/payloads(18-112, 19-113, 20-114, 21-120, 22-121, 23-122) to establishantibody-drug conjugates (ADCs).

FIG. 33 illustrates a CTG assay wherein ROR+(JeKo-1) cells wereincubated with the ADCs generated as described in FIG. 32 in serialthree-fold dilutions and cell viability was assessed after 72 hours.

FIG. 34 illustrates nucleotide and amino acid sequences for anti-ROR-1antibodies ATX-P-875, ATX-P-885, and ATX-P-890.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. All patents, applications, published applications and otherpublications referenced herein are incorporated by reference in theirentirety unless stated otherwise. In the event that there are aplurality of definitions for a term herein, those in this sectionprevail unless stated otherwise.

As used herein, a “conjugate” is a compound that comprises two or moresubstances (such as an antibody, a linker moiety and/or a drug moiety)joined together by chemical bonds. Examples of conjugates includeantibody-drug conjugates (which may optionally include a linker moiety),drug-linker conjugates, and antibody-linker conjugates. An“immunoconjugate” is a conjugate that comprise an immunologicalsubstance such as an antibody.

As used herein, an “antibody” (Ab) is a protein made by the immunesystem, or a synthetic variant thereof, that binds to specific sites oncells or tissues. An “antigen-binding fragment” (Fab) is a portion of anantibody that binds to a specific antigen. Monoclonal antibodies are atype of synthetic antibody. In cancer treatment, monoclonal antibodiesmay kill cancer cells directly, they may block development of tumorblood vessels, or they may help the immune system kill cancer cells.

Whenever a group is described as being “optionally substituted” thatgroup may be unsubstituted or substituted with one or more of theindicated substituents. Likewise, when a group is described as being“unsubstituted or substituted” if substituted, the substituent(s) may beselected from one or more the indicated substituents. If no substituentsare indicated, it is meant that the indicated “optionally substituted”or “substituted” group may be substituted with one or more group(s)individually and independently selected from alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, aryl(alkyl),cycloalkyl(alkyl), heteroaryl(alkyl), heterocyclyl(alkyl), hydroxy,alkoxy, acyl, cyano, halogen, thiocarbonyl, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido,N-sulfonamido, C-carboxy, O-carboxy, nitro, sulfenyl, sulfinyl,sulfonyl, haloalkyl, haloalkoxy, an amino, a mono-substituted aminegroup, a di-substituted amine group, a mono-substituted amine(alkyl) anda di-substituted amine(alkyl).

As used herein, “C_(a) to C_(b)” in which “a” and “b” are integers referto the number of carbon atoms in a group. The indicated group cancontain from “a” to “b”, inclusive, carbon atoms. Thus, for example, a“C₁ to C₄ alkyl” group refers to all alkyl groups having from 1 to 4carbons, that is, CH₃—, CH₃CH₂—, CH₃CH₂CH₂—, (CH₃)₂CH—, CH₃CH₂CH₂CH₂—,CH₃CH₂CH(CH₃)— and (CH₃)₃C—. If no “a” and “b” are designated, thebroadest range described in these definitions is to be assumed.

If two “R” groups are described as being “taken together” the R groupsand the atoms they are attached to can form a cycloalkyl, cycloalkenyl,aryl, heteroaryl or heterocycle. For example, without limitation, ifortho R¹ and R² substituents on a phenyl ring are indicated to be—O—(CR⁵R⁶)_(m)—O— such that R¹ and R² “taken together” form a ring, itmeans that the —O—(CR⁵R⁶)_(m)—O— is covalently bonded to the phenyl ringat the R¹ and R² positions to form a heterocyclic ring:

As used herein, the term “alkyl” refers to a fully saturated aliphatichydrocarbon group. The alkyl moiety may be branched or straight chain.Examples of branched alkyl groups include, but are not limited to,iso-propyl, sec-butyl, t-butyl and the like. Examples of straight chainalkyl groups include, but are not limited to, methyl, ethyl, n-propyl,n-butyl, n-pentyl, n-hexyl, n-heptyl and the like. The alkyl group mayhave 1 to 30 carbon atoms (whenever it appears herein, a numerical rangesuch as “1 to 30” refers to each integer in the given range; e.g., “1 to30 carbon atoms” means that the alkyl group may consist of 1 carbonatom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 30carbon atoms, although the present definition also covers the occurrenceof the term “alkyl” where no numerical range is designated). The alkylgroup may also be a medium size alkyl having 1 to 12 carbon atoms. Thealkyl group could also be a lower alkyl having 1 to 6 carbon atoms. Analkyl group may be substituted or unsubstituted. An alkyl group istypically monovalent unless the context indicates otherwise. Forexample, those skilled in the art recognize that C₁-C₆ alkyl is bivalentin the following formula: —(C₁-C₆ alkyl)-X².

As used herein, the term “alkylene” refers to a bivalent fully saturatedstraight chain aliphatic hydrocarbon group. Examples of alkylene groupsinclude, but are not limited to, methylene, ethylene, propylene,butylene, pentylene, hexylene, heptylene and octylene. An alkylene groupmay be represented by

, followed by the number of carbon atoms, followed by a “*”. Forexample,

to represent ethylene. The alkylene group may have 1 to 30 carbon atoms(whenever it appears herein, a numerical range such as “1 to 30” refersto each integer in the given range; e.g., “1 to 30 carbon atoms” meansthat the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3carbon atoms, etc., up to and including 30 carbon atoms, although thepresent definition also covers the occurrence of the term “alkylene”where no numerical range is designated). The alkylene group may also bea medium size alkyl having 1 to 12 carbon atoms. The alkylene groupcould also be a lower alkyl having 1 to 4 carbon atoms. An alkylenegroup may be substituted or unsubstituted. For example, a lower alkylenegroup can be substituted by replacing one or more hydrogen of the loweralkylene group and/or by substituting both hydrogens on the same carbonwith a C₃₋₆ monocyclic cycloalkyl group

The term “alkenyl” used herein refers to a monovalent straight orbranched chain radical of from two to twenty carbon atoms containing acarbon double bond(s) including, but not limited to, 1-propenyl,2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl and the like. Analkenyl group may be unsubstituted or substituted.

The term “alkynyl” used herein refers to a monovalent straight orbranched chain radical of from two to twenty carbon atoms containing acarbon triple bond(s) including, but not limited to, 1-propynyl,1-butynyl, 2-butynyl and the like. An alkynyl group may be unsubstitutedor substituted.

The term “halogen atom” or “halogen” as used herein, means any one ofthe radio-stable atoms of column 7 of the Periodic Table of theElements, such as, fluorine, chlorine, bromine and iodine.

As used herein, “haloalkyl” refers to an alkyl group in which one ormore of the hydrogen atoms are replaced by a halogen (e.g.,mono-haloalkyl, di-haloalkyl, tri-haloalkyl and polyhaloalkyl). Suchgroups include but are not limited to, chloromethyl, fluoromethyl,difluoromethyl, trifluoromethyl, 1-chloro-2-fluoromethyl,2-fluoroisobutyl and pentafluoroethyl. A haloalkyl may be substituted orunsubstituted.

As used herein, “haloalkenyl” refers to an alkenyl group in which one ormore of the hydrogen atoms are replaced by a halogen (e.g.,mono-haloalkenyl, di-haloalkenyl, tri-haloalkenyl and polyhaloalkenyl).

As used herein, “haloalkynyl” refers to an alkynyl group in which one ormore of the hydrogen atoms are replaced by a halogen (e.g.,mono-haloalkynyl, di-haloalkynyl, tri-haloalkynyl and polyhaloalkynyl).

As used herein, “haloalkoxy” refers to an alkoxy group in which one ormore of the hydrogen atoms are replaced by a halogen (e.g.,mono-haloalkoxy, di-haloalkoxy and tri-haloalkoxy). Such groups includebut are not limited to, chloromethoxy, fluoromethoxy, difluoromethoxy,trifluoromethoxy, 1-chloro-2-fluoromethoxy and 2-fluoroisobutoxy. Ahaloalkoxy may be substituted or unsubstituted.

Where the number of substituents is not specified (e.g. haloalkyl,haloalkenyl, haloalkynyl), there may be one or more substituentspresent. For example, “haloalkyl” may include one or more of the same ordifferent halogens. As another example, “C₁-C₃ alkoxyphenyl” may includeone or more of the same or different alkoxy groups containing one, twoor three atoms.

As used herein, a radical indicates species with a single, unpairedelectron such that the species containing the radical can be covalentlybonded to another species. Hence, in this context, a radical is notnecessarily a free radical. Rather, a radical indicates a specificportion of a larger molecule. The term “radical” can be usedinterchangeably with the term “group.”

The term “pharmaceutically acceptable salt” refers to a salt of acompound that does not cause significant irritation to an organism towhich it is administered and does not abrogate the biological activityand properties of the compound. In some embodiments, the salt is an acidaddition salt of the compound. Pharmaceutical salts can be obtained byreacting a compound with inorganic acids such as hydrohalic acid (e.g.,hydrochloric acid or hydrobromic acid), a sulfuric acid, a nitric acidand a phosphoric acid (such as 2,3-dihydroxypropyl dihydrogenphosphate). Pharmaceutical salts can also be obtained by reacting acompound with an organic acid such as aliphatic or aromatic carboxylicor sulfonic acids, for example formic, acetic, succinic, lactic, malic,tartaric, citric, ascorbic, nicotinic, methanesulfonic, ethanesulfonic,p-toluensulfonic, trifluoroacetic, benzoic, salicylic, 2-oxopentanedioicor naphthalenesulfonic acid. Pharmaceutical salts can also be obtainedby reacting a compound with a base to form a salt such as an ammoniumsalt, an alkali metal salt, such as a sodium, a potassium or a lithiumsalt, an alkaline earth metal salt, such as a calcium or a magnesiumsalt, a salt of a carbonate, a salt of a bicarbonate, a salt of organicbases such as dicyclohexylamine, N-methyl-D-glucamine,tris(hydroxymethyl)methylamine, C₁-C₇ alkylamine, cyclohexylamine,triethanolamine, ethylenediamine and salts with amino acids such asarginine and lysine. For compounds of Formula (I), those skilled in theart understand that when a salt is formed by protonation of anitrogen-based group (for example, NH₂), the nitrogen-based group can beassociated with a positive charge (for example, NH₂ can become NH₃ ⁺)and the positive charge can be balanced by a negatively chargedcounterion (such as Cl^(−).)

It is understood that, in any compound described herein having one ormore chiral centers, if an absolute stereochemistry is not expresslyindicated, then each center may independently be of R-configuration orS-configuration or a mixture thereof. Thus, the compounds providedherein may be enantiomerically pure, enantiomerically enriched, racemicmixture, diastereomerically pure, diastereomerically enriched or astereoisomeric mixture. In addition, it is understood that, in anycompound described herein having one or more double bond(s) generatinggeometrical isomers that can be defined as E or Z, each double bond mayindependently be E or Z a mixture thereof. Likewise, it is understoodthat, in any compound described, all tautomeric forms are also intendedto be included.

It is to be understood that where compounds disclosed herein haveunfilled valencies, then the valencies are to be filled with hydrogensor isotopes thereof, e.g., hydrogen-1 (protium) and hydrogen-2(deuterium).

It is understood that the compounds described herein can be labeledisotopically. Substitution with isotopes such as deuterium may affordcertain therapeutic advantages resulting from greater metabolicstability, such as, for example, increased in vivo half-life or reduceddosage requirements. Each chemical element as represented in a compoundstructure may include any isotope of said element. For example, in acompound structure a hydrogen atom may be explicitly disclosed orunderstood to be present in the compound. At any position of thecompound that a hydrogen atom may be present, the hydrogen atom can beany isotope of hydrogen, including but not limited to hydrogen-1(protium) and hydrogen-2 (deuterium). Thus, reference herein to acompound encompasses all potential isotopic forms unless the contextclearly dictates otherwise.

It is understood that the methods and combinations described hereininclude crystalline forms (also known as polymorphs, which include thedifferent crystal packing arrangements of the same elemental compositionof a compound), amorphous phases, salts, solvates and hydrates. In someembodiments, the compounds described herein exist in solvated forms withpharmaceutically acceptable solvents such as water, ethanol or the like.In other embodiments, the compounds described herein exist in unsolvatedform. Solvates contain either stoichiometric or non-stoichiometricamounts of a solvent, and may be formed during the process ofcrystallization with pharmaceutically acceptable solvents such as water,ethanol or the like. Hydrates are formed when the solvent is water oralcoholates are formed when the solvent is alcohol. In addition, thecompounds provided herein can exist in unsolvated as well as solvatedforms. In general, the solvated forms are considered equivalent to theunsolvated forms for the purposes of the compounds and methods providedherein.

Where a range of values is provided, it is understood that the upper andlower limit, and each intervening value between the upper and lowerlimit of the range is encompassed within the embodiments.

Terms and phrases used in this application, and variations thereof,especially in the appended claims, unless otherwise expressly stated,should be construed as open ended as opposed to limiting. As examples ofthe foregoing, the term ‘including’ should be read to mean ‘including,without limitation,’ ‘including but not limited to,’ or the like; theterm ‘comprising’ as used herein is synonymous with ‘including,’‘containing,’ or ‘characterized by,’ and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps; theterm ‘having’ should be interpreted as ‘having at least;’ the term‘includes’ should be interpreted as ‘includes but is not limited to;’the term ‘example’ is used to provide exemplary instances of the item indiscussion, not an exhaustive or limiting list thereof; and use of termslike ‘preferably,’ ‘preferred,’ ‘desired,’ or ‘desirable,’ and words ofsimilar meaning should not be understood as implying that certainfeatures are critical, essential, or even important to the structure orfunction, but instead as merely intended to highlight alternative oradditional features that may or may not be utilized in a particularembodiment. In addition, the term “comprising” is to be interpretedsynonymously with the phrases “having at least” or “including at least”.When used in the context of a compound, composition or device, the term“comprising” means that the compound, composition or device includes atleast the recited features or components, but may also includeadditional features or components.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity. The indefinite article “a” or “an” does not exclude aplurality. The mere fact that certain measures are recited in mutuallydifferent dependent claims does not indicate that a combination of thesemeasures cannot be used to advantage. Any reference signs in the claimsshould not be construed as limiting the scope.

Compounds

Various embodiments disclosed herein relate to a compound of Formula(IV), or a pharmaceutically acceptable salt thereof, having thestructure:

In various embodiments, R¹ and R² in Formula (IV) are each individuallyselected from the group consisting of hydrogen, halogen, —CN, —OR⁵,—NR⁵R⁶, a substituted or an unsubstituted C₁-C₆ alkyl, a substituted oran unsubstituted C₁-C₆ haloalkyl, a substituted or an unsubstituted—O—(C₁-C₆ alkyl), a substituted or an unsubstituted —O—(C₁-C₆haloalkyl), —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃, or a substituted or anunsubstituted —O—(CR⁵R⁶)_(m)—O— such that R¹ and R² taken together forma ring. In an embodiment, at least one of R¹ and R² is hydrogen. In anembodiment, at least one of R¹ and R² is halogen. For example, in anembodiment, at least one of R¹ and R² is fluoro. In an embodiment, atleast one of R¹ and R² is —CN. In an embodiment, at least one of R¹ andR² is —OR⁵, wherein R⁵ is hydrogen, halogen, a substituted or anunsubstituted C₁-C₆ alkyl, a substituted or an unsubstituted C₁-C₆haloalkyl, or —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃. For example, in anembodiment, at least one of R¹ and R² is methoxy.

In an embodiment, at least one of R¹ and R² in Formula (IV) is —NR⁵R⁶,wherein R⁵ and R⁶ are each individually hydrogen, halogen, a substitutedor an unsubstituted C₁-C₆ alkyl, a substituted or an unsubstituted C₁-C₆haloalkyl, or —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃. In an embodiment, at leastone of R¹ and R² is a substituted or an unsubstituted C₁-C₆ alkyl. Forexample, in an embodiment, at least one of R¹ and R² is methyl. In anembodiment, at least one of R¹ and R² is a substituted or anunsubstituted C₁-C₆ haloalkyl. For example, in an embodiment, at leastone of R¹ and R² is difluoromethyl. In an embodiment, at least one of R¹and R² is a substituted or an unsubstituted —O—(C₁-C₆ alkyl). Forexample, in an embodiment, at least one of R¹ and R² is methoxy. In anembodiment, at least one of R¹ and R² is —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃.In an embodiment, R¹ and R² are a substituted or an unsubstituted—O—(CR⁵R⁶)_(m)—O— such that R¹ and R² taken together form a ring inwhich the ends of the —O—(CR⁵R⁶)_(m)—O— are covalently bonded to thephenyl ring at the R¹ and R² positions of Formula (IV) to form aheterocyclic ring.

In an embodiment, one of R¹ and R² in Formula (IV) is hydrogen and theother of R¹ and R² is halogen. In an embodiment, one of R¹ and R² ishydrogen and the other of R¹ and R² is a substituted or an unsubstitutedC₁-C₆ alkyl. In an embodiment, one of R¹ and R² is hydrogen and theother of R¹ and R² is a substituted or an unsubstituted C₁-C₆ haloalkyl.In an embodiment, one of R¹ and R² is hydrogen and the other of R¹ andR² is a substituted or an unsubstituted —O—(C₁-C₆ alkyl). In anembodiment, both R¹ and R² are hydrogen. In an embodiment, neither R¹nor R² is hydrogen.

In an embodiment, one of R¹ and R² in Formula (IV) is halogen and theother of R¹ and R² is a substituted or an unsubstituted C₁-C₆ alkyl. Inan embodiment, one of R¹ and R² is halogen and the other of R¹ and R² isa substituted or an unsubstituted C₁-C₆ haloalkyl. In an embodiment, oneof R¹ and R² is halogen and the other of R¹ and R² is a substituted oran unsubstituted —O—(C₁-C₆ alkyl). In an embodiment, both R¹ and R² areindependently halogen. In an embodiment, neither R¹ nor R² is halogen.

In an embodiment, one of R¹ and R² in Formula (IV) is a substituted oran unsubstituted C₁-C₆ alkyl and the other of R¹ and R² is a substitutedor an unsubstituted C₁-C₆ haloalkyl. In an embodiment, one of R¹ and R²is a substituted or an unsubstituted C₁-C₆ alkyl and the other of R¹ andR² is a substituted or an unsubstituted —O—(C₁-C₆ alkyl). In anembodiment, both R¹ and R² are independently a substituted or anunsubstituted C₁-C₆ alkyl. In an embodiment, neither R¹ nor R² is asubstituted or an unsubstituted C₁-C₆ alkyl.

In an embodiment, one of R¹ and R² in Formula (IV) is a substituted oran unsubstituted C₁-C₆ haloalkyl and the other of R¹ and R² is asubstituted or an unsubstituted —O—(C₁-C₆ alkyl). In an embodiment, bothR¹ and R² are independently a substituted or an unsubstituted C₁-C₆haloalkyl. In an embodiment, neither R¹ nor R² is a substituted or anunsubstituted C₁-C₆ haloalkyl.

In an embodiment, one of R¹ and R² in Formula (IV) is a substituted oran unsubstituted —O—(C₁-C₆ alkyl). In an embodiment, both R¹ and R² areindependently a substituted or an unsubstituted —O—(C₁-C₆ alkyl). In anembodiment, neither R¹ nor R² is a substituted or an unsubstituted—O—(C₁-C₆ alkyl). In an embodiment, R¹ and R² are a substituted or anunsubstituted —O—(CR⁵R⁶)_(m)—O— such that R¹ and R² taken together forma ring. In various embodiments, R¹ and R² are each individually selectedfrom the group consisting of hydrogen, fluoro, methoxy, methyl,difluoromethyl, and —O—(CH₂)—O— such that R¹ and R² taken together forma ring.

In various embodiments, R³ in Formula (IV) is hydrogen or a substitutedor an unsubstituted C₁-C₆ alkyl, a substituted or an unsubstituted C₁-C₆haloalkyl, or —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃, where each Y isindividually H or halogen. In an embodiment, R³ is hydrogen. In anembodiment, R³ is a substituted or an unsubstituted C₁-C₆ alkyl. Forexample, in an embodiment, R³ is methyl. In an embodiment, R³ is asubstituted or an unsubstituted C₁-C₆ haloalkyl. In an embodiment, R³ is—[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃, where each Y is individually H orhalogen.

In various embodiments, R⁴ in Formula (IV) is hydrogen, a substituted oran unsubstituted —(C₁-C₆ alkyl)-X², a substituted or an unsubstituted—(C₁-C₆ haloalkyl)-X², a substituted or an unsubstituted —(C₁-C₆alkenyl)-X², a substituted or an unsubstituted —(C₁-C₆ haloalkenyl)-X²,a substituted or an unsubstituted —(C₁-C₆ alkynyl)-X², a substituted oran unsubstituted —(C₁-C₆ haloalkynyl)-X², where X² is —OH, —SH, or—NR⁵R⁶. In an embodiment, R⁴ is hydrogen. In an embodiment, R⁴ is asubstituted or an unsubstituted —(C₁-C₆ alkyl)-X². In an embodiment, R⁴is a substituted or an unsubstituted —(C₁-C₆ haloalkyl)-X². In anembodiment, R⁴ is a substituted or an unsubstituted —(C₁-C₆ alkenyl)-X².In an embodiment, R⁴ is a substituted or an unsubstituted —(C₁-C₆haloalkenyl)-X². In an embodiment, R⁴ is a substituted or anunsubstituted —(C₁-C₆ alkynyl)-X². In an embodiment, R⁴ is a substitutedor an unsubstituted —(C₁-C₆ haloalkynyl)-X².

In various embodiments, X¹ in Formula (IV) is —O—, —S(O_(n6))—, —NH—,—O—(C═O)—, —NH—(C═O)—, —NH—(C═O)—O—, —NH—(C═O)—NH—, or —NH—S(O_(n6))—,where n⁶ is 0, 1 or 2. In an embodiment, X¹ is —O—. In an embodiment, X¹is —S(O_(n6))—. In an embodiment, X¹ is —NH—. In an embodiment, X¹ is—O—(C═O)—. In an embodiment, X¹ is —NH—(C═O)—. In an embodiment, X¹ is—NH—(C═O)—O—. In an embodiment, X¹ is —NH—(C═O)—NH—. In an embodiment,X¹ is —NH—S(O_(n6))—.

In various embodiments, X² in Formula (IV) is —OH, —SH, or —NR⁵R⁶, whereR⁵ and R⁶ are each individually hydrogen, halogen or a substituted or anunsubstituted C₁-C₆ alkyl, C₁-C₆ haloalkyl,—[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃. In an embodiment, X² is —OH. In anembodiment, X² is —SH. In an embodiment, X² is —NR⁵R⁶.

In various embodiments, R⁵ and R⁶ in Formula (IV) are each individuallyhydrogen, halogen, a substituted or an unsubstituted C₁-C₆ alkyl, asubstituted or an unsubstituted C₁-C₆ haloalkyl, or—[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃, where each Y is individually H or halogenand variables p, q and t are as described elsewhere herein. In anembodiment, R⁵ and R⁶ are each individually hydrogen or a substituted oran unsubstituted C₁-C₆ alkyl. In an embodiment, R⁵ and R⁶ are bothhydrogen. In an embodiment, R⁵ and R⁶ are each individually asubstituted or an unsubstituted C₁-C₆ alkyl.

In various embodiments, R⁷ in Formula (IV) is H, —COR⁸, —CO₂R⁸, or—(CO)—NHR⁸, wherein R⁸ is described elsewhere herein. In an embodiment,R⁷ is H. In an embodiment, R⁷ is —COR⁸. In an embodiment, R⁷ is —CO₂R⁸.In an embodiment, R⁷ is —(CO)—NHR⁸.

In various embodiments, R⁸ in Formula (IV) is a substituted or anunsubstituted C₁-C₆ alkyl, a substituted or an unsubstituted C₁-C₆haloalkyl, or —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃, where the variables p, q, tand Y are described elsewhere herein. In an embodiment, R⁸ is asubstituted or an unsubstituted C₁-C₆ alkyl. In an embodiment, R⁸ is asubstituted or an unsubstituted C₁-C₆ haloalkyl. In an embodiment, R⁸ isa —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃.

In various embodiments, m in Formula (IV) is 1 or 2. In an embodiment, mis 1. In another embodiment, m is 2.

In various embodiments, n⁴ and n⁵ in Formula (IV) are each individually0, 1 or 2, with the proviso that n⁴ and n⁵ are not both 0. In anembodiment, n⁴ and n⁵ are both 1. In an embodiment, n⁴ is 0 and n⁵ is 1.In an embodiment, n⁴ is 0 and n⁵ is 2. In an embodiment, n⁴ is 1 and n⁵is 0. In an embodiment, n⁴ is 2 and n⁵ is 0.

In various embodiments, n⁶ in Formula (IV) is 0, 1 or 2. In anembodiment, n⁶ is 0, in which case X¹ is —S— or —NH—S—. In anembodiment, n⁶ is 1, in which case X¹ is —S(═O)— or —NH—S(═O)—. In anembodiment, n⁶ is 2, in which case X¹ is —S(═O)₂— or —NH—S(═O)₂—.

In various embodiments, each Y in Formula (IV) is individually H orhalogen. In an embodiment, each Y is hydrogen. In an embodiment, —CY₂ isCH₂. In an embodiment, —CY₃ is CH₃. In an embodiment, —CY₃ is CHF₂. Inan embodiment, —CH₂F is CH₃. In an embodiment, —CY₃ is CF₃.

In various embodiments, each p in Formula (IV) is individually 1, 2, 3,4, 5, or 6. In an embodiment, p is 1. In an embodiment, p is 2.

In various embodiments, each q in Formula (IV) is individually 0, 1, 2,3, 4, 5, or 6. In an embodiment, q is 1. In an embodiment, q is 2.

In various embodiments, each t in Formula (IV) is individually 1, 2, 3,4, 5, or 6. In an embodiment, t is 1. In an embodiment, p is t.

In various embodiments, Formula (IV) does not represent deruxtecan orexatecan.

In various embodiments, the compound of Formula (IV) is represented byFormula (IVa):

In Formula (IVa), the variables are the same as defined elsewhere hereinfor Formula (IV).

In various embodiments, the compound of Formula (IV) is represented byFormula (IVb):

In Formula (IVb), the variables are the same as defined elsewhere hereinfor Formula (IV).

In various embodiments, the compound of Formula (IV) is represented byFormula (IVc):

In Formula (IVc), the variables are the same as defined elsewhere hereinfor Formula (IV).

In various embodiments, the compound of Formula (IV) is represented by astructure selected from the following, or a pharmaceutically acceptablesalt thereof:

Conjugates

Various embodiments disclosed herein relate to a conjugate of Formula(III), having the structure:

Mi-L²-L³-L⁴-L⁵-L⁶-L⁷-D   (III)

In various embodiments, Mi in Formula (III) is

D is a drug moiety and -L²-L³-L⁴-L⁵-L⁶-L⁷- is a linker that connects Mito D.

In various embodiments, L² in Formula (III) is absent,

where Z¹ and Z² are each individually hydrogen, halogen, NO₂, —O—(C₁-C₆alkyl), or C₁-C₆ alkyl. In an embodiment, L² in Formula (III) is absent.In an embodiment, L² in Formula (III) is

In an embodiment, L² in Formula (III) is

In various embodiments, Z¹ and Z² in Formula (III) are each individuallyhydrogen, halogen, NO₂, —O—(C₁-C₆ alkyl), or C₁-C₆ alkyl. In anembodiment, at least one of Z¹ and Z² is hydrogen. In an embodiment, atleast one of Z¹ and Z² is halogen. In an embodiment, at least one of Z¹and Z² is NO₂. In an embodiment, at least one of Z¹ and Z² is —O—(C₁-C₆alkyl). For example, in an embodiment, at least one of Z¹ and Z² ismethoxy. In an embodiment, at least one of Z¹ and Z² is C₁-C₆ alkyl. Forexample, in an embodiment, at least one of Z¹ and Z² is methyl.

In various embodiments, L³ in Formula (III) is —(CH₂)n¹-C(═O)— or—(CH₂CH₂O)n¹-(CH₂)n¹C(═O)—, where n¹ are independently integers of 0 to12. In an embodiment, L³ is —(CH₂)n¹-C(═O)—. For example, in anembodiment, L³ is —C(═O)—. In an embodiment, L³ is—(CH₂CH₂O)n¹-(CH₂)n¹C(═O)—. For example, in an embodiment, L³ is—CH₂C(═O)—. In embodiment, n¹ is an integer of 1 to 12, such as 1 to 6or 1 to 3.

In various embodiments, L⁴ in Formula (III) is a tetrapeptide residue.For example, in an embodiment, L⁴ is a tetrapeptide residue selectedfrom SEQ ID NO: 43 GGFG (gly-gly-phe-gly), SEQ ID NO: 44 EGGF(glu-gly-gly-phe), SEQ ID NO: 45 SGGF (ser-gly-gly-phe), and SEQ ID NO46: KGGF (lys-gly-gly-phe).

In various embodiments, L⁵ in Formula (III) is absent or—[NH(CH₂)n²]n³-, where n² is an integer of 0 to 6 and n³ is an integerof 0 to 2. In an embodiment, L⁵ is absent. In an embodiment, L⁵ is—[NH(CH₂)n²]n³-. For example, in an embodiment, L⁵ is —NH—. In anotherembodiment, L⁵ is —NHCH₂—.

In various embodiments, L⁶ in Formula (III) is absent or

In an embodiment, L⁶ is absent. In another embodiment, L⁶ is

In various embodiments, L⁷ in Formula (III) is absent,

In an embodiment, L⁷ is absent.

In an embodiment, L⁷ is

In an embodiment, L⁷ is

In an embodiment, L⁷ is

In various embodiments, D in the conjugate of Formula (III) is a drugmoiety as described herein (e.g., under the heading “Drug Moieties”below). In an embodiment, D is a cytotoxic anti-cancer drug moiety.

In various embodiments, the conjugate of Formula (III) is represented bya structure selected from the following, for which Z¹ and Z² are eachindividually selected from hydrogen, fluoro, chloro, —NO₂, and —OCH₃:

Drug Moieties

In various embodiments, D in the immunoconjugate of Formula (I) or inthe conjugate of Formula (III) is a drug moiety. The drug moiety may beany compound of the Formula (IV) as described herein (e.g., as describedabove under the heading “Compounds”), with appropriate modification sothat the linker -L²-L³-L⁴-L⁵-L⁶-L⁷- connects to D. For example, invarious embodiments the drug moiety D is a compound of Formula (II)having the structure:

Those skilled in the art will appreciate that the compound of Formula(II) connects to the linker -L²-L³-L⁴-L⁵-L⁶-L⁷- via R⁴ (when defined toinclude X² and thus R⁹) or via R⁷ as described below.

In various embodiments, R¹ and R² in Formula (II) are each individuallyselected from the group consisting of hydrogen, halogen, —CN, —OR⁵,—NR⁵R⁶, a substituted or an unsubstituted C₁-C₆ alkyl, a substituted oran unsubstituted C₁-C₆ haloalkyl, a substituted or an unsubstituted—O—(C₁-C₆ alkyl), a substituted or an unsubstituted —O—(C₁-C₆haloalkyl), —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃, or a substituted or anunsubstituted —O—(CR⁵R⁶)_(m)—O— such that R¹ and R² taken together forma ring. In an embodiment, at least one of R¹ and R² is hydrogen. In anembodiment, at least one of R¹ and R² is halogen. For example, in anembodiment, at least one of R¹ and R² is fluoro. In an embodiment, atleast one of R¹ and R² is —CN. In an embodiment, at least one of R¹ andR² is —OR⁵, wherein R⁵ is hydrogen, halogen, a substituted or anunsubstituted C₁-C₆ alkyl, a substituted or an unsubstituted C₁-C₆haloalkyl, or —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃. For example, in anembodiment, at least one of R¹ and R² is methoxy.

In an embodiment, at least one of R¹ and R² in Formula (II) is —NR⁵R⁶,wherein R⁵ and R⁶ are each individually hydrogen, halogen, a substitutedor an unsubstituted C₁-C₆ alkyl, a substituted or an unsubstituted C₁-C₆haloalkyl, or —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃. In an embodiment, at leastone of R¹ and R² is a substituted or an unsubstituted C₁-C₆ alkyl. Forexample, in an embodiment, at least one of R¹ and R² is methyl. In anembodiment, at least one of R¹ and R² is a substituted or anunsubstituted C₁-C₆ haloalkyl. For example, in an embodiment, at leastone of R¹ and R² is difluoromethyl. In an embodiment, at least one of R¹and R² is a substituted or an unsubstituted —O—(C₁-C₆ alkyl). Forexample, in an embodiment, at least one of R¹ and R² is methoxy. In anembodiment, at least one of R¹ and R² is —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃.In an embodiment, R¹ and R² are a substituted or an unsubstituted—O—(CR⁵R⁶)_(m)—O— such that R¹ and R² taken together form a ring inwhich the ends of the —O—(CR⁵R⁶)_(m)—O— are covalently bonded to thephenyl ring at the R¹ and R² positions of Formula (IV) to form aheterocyclic ring.

In an embodiment, one of R¹ and R² in Formula (II) is hydrogen and theother of R¹ and R² is halogen. In an embodiment, one of R¹ and R² ishydrogen and the other of R¹ and R² is a substituted or an unsubstitutedC₁-C₆ alkyl. In an embodiment, one of R¹ and R² is hydrogen and theother of R¹ and R² is a substituted or an unsubstituted C₁-C₆ haloalkyl.In an embodiment, one of R¹ and R² is hydrogen and the other of R¹ andR² is a substituted or an unsubstituted —O—(C₁-C₆ alkyl). In anembodiment, both R¹ and R² are hydrogen. In an embodiment, neither R¹nor R² is hydrogen.

In an embodiment, one of R¹ and R² in Formula (II) is halogen and theother of R¹ and R² is a substituted or an unsubstituted C₁-C₆ alkyl. Inan embodiment, one of R¹ and R² is halogen and the other of R¹ and R² isa substituted or an unsubstituted C₁-C₆ haloalkyl. In an embodiment, oneof R¹ and R² is halogen and the other of R¹ and R² is a substituted oran unsubstituted —O—(C₁-C₆ alkyl). In an embodiment, both R¹ and R² areindependently halogen. In an embodiment, neither R¹ nor R² is halogen.

In an embodiment, one of R¹ and R² in Formula (II) is a substituted oran unsubstituted C₁-C₆ alkyl and the other of R¹ and R² is a substitutedor an unsubstituted C₁-C₆ haloalkyl. In an embodiment, one of R¹ and R²is a substituted or an unsubstituted C₁-C₆ alkyl and the other of R¹ andR² is a substituted or an unsubstituted —O—(C₁-C₆ alkyl). In anembodiment, both R¹ and R² are independently a substituted or anunsubstituted C₁-C₆ alkyl. In an embodiment, neither R¹ nor R² is asubstituted or an unsubstituted C₁-C₆ alkyl.

In an embodiment, one of R¹ and R² in Formula (II) is a substituted oran unsubstituted C₁-C₆ haloalkyl and the other of R¹ and R² is asubstituted or an unsubstituted —O—(C₁-C₆ alkyl). In an embodiment, bothR¹ and R² are independently a substituted or an unsubstituted C₁-C₆haloalkyl. In an embodiment, neither R¹ nor R² is a substituted or anunsubstituted C₁-C₆ haloalkyl.

In an embodiment, one of R¹ and R² in Formula (II) is a substituted oran unsubstituted —O—(C₁-C₆ alkyl). In an embodiment, both R¹ and R² areindependently a substituted or an unsubstituted —O—(C₁-C₆ alkyl). In anembodiment, neither R¹ nor R² is a substituted or an unsubstituted—O—(C₁-C₆ alkyl). In an embodiment, R¹ and R² are a substituted or anunsubstituted —O—(CR⁵R⁶)_(m)—O— such that R¹ and R² taken together forma ring. In various embodiments, R¹ and R² are each individually selectedfrom the group consisting of hydrogen, fluoro, methoxy, methyl,difluoromethyl, and —O—(CH₂)—O— such that R¹ and R² taken together forma ring.

In various embodiments, R³ in Formula (II) is hydrogen or a substitutedor an unsubstituted C₁-C₆ alkyl, a substituted or an unsubstituted C₁-C₆haloalkyl, or —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃, where each Y isindividually H or halogen. In an embodiment, R³ is hydrogen. In anembodiment, R³ is a substituted or an unsubstituted C₁-C₆ alkyl. Forexample, in an embodiment, R³ is methyl. In an embodiment, R³ is asubstituted or an unsubstituted C₁-C₆haloalkyl. In an embodiment, R³ is—[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃, where each Y is individually H orhalogen.

In various embodiments, R⁴ in Formula (II) is hydrogen, a substituted oran unsubstituted —(C₁-C₆ alkyl)-X², a substituted or an unsubstituted—(C₁-C₆ haloalkyl)-X², a substituted or an unsubstituted —(C₁-C₆alkenyl)-X², a substituted or an unsubstituted —(C₁-C₆ haloalkenyl)-X²,a substituted or an unsubstituted —(C₁-C₆ alkynyl)-X², a substituted oran unsubstituted —(C₁-C₆ haloalkynyl)-X², where X² is —OR⁹, —SR⁹, or—NHR⁹ and R⁹ is H, absent, —COR⁸, —CO₂R⁸, —(CO)—NHR⁸, L⁴, L⁵, L⁶, or L⁷.In an embodiment, R⁴ is hydrogen, in which case X² is not present andthe compound of Formula (II) connects to the linker -L²-L³-L⁴-L⁵-L⁶-L⁷-via R⁷ as described in greater detail below.

In other embodiments, the compound of Formula (II) connects to thelinker -L²-L³-L⁴-L⁵-L⁶-L⁷- via R⁴ when R⁴ includes X² and R⁹ is L⁴, L⁵,L⁶, or L⁷. In an embodiment R⁴ is a substituted or an unsubstituted—(C₁-C₆ alkyl)-X². In an embodiment, R⁴ is a substituted or anunsubstituted —(C₁-C₆ haloalkyl)-X². In an embodiment, R⁴ is asubstituted or an unsubstituted —(C₁-C₆ alkenyl)-X². In an embodiment,R⁴ is a substituted or an unsubstituted —(C₁-C₆ haloalkenyl)-X². In anembodiment, R⁴ is a substituted or an unsubstituted —(C₁-C₆ alkynyl)-X².In an embodiment, R⁴ is a substituted or an unsubstituted —(C₁-C₆haloalkynyl)-X². In each such embodiment in which R⁴ includes X², theoption for R⁹ to be L⁴, L⁵, L⁶, or L⁷ is provided, thus providing theoption to thereby connect the compound of Formula (II) to the linker-L²-L³-L⁴-L⁵-L⁶-L⁷- via R⁴.

In various embodiments, X¹ in Formula (II) is —O—, —S(O_(n6))—, —NH—,—O—(C═O)—, —NH—(C═O)—, —NH—(C═O)—O—, —NH—(C═O)—NH—, or —NH—S(O_(n6))—,where n⁶ is 0, 1 or 2. In an embodiment, X¹ is —O—. In an embodiment, X¹is —S(O_(n6))—. In an embodiment, X¹ is —NH—. In an embodiment, X¹ is—O—(C═O)—. In an embodiment, X¹ is —NH—(C═O)—. In an embodiment, X¹ is—NH—(C═O)—O—. In an embodiment, X¹ is —NH—(C═O)—NH—. In an embodiment,X¹ is —NH—S(O_(n6))—.

In various embodiments, R⁵ and R⁶ in Formula (II) are each individuallyhydrogen, halogen, a substituted or an unsubstituted C₁-C₆ alkyl, C₁-C₆haloalkyl, or —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃, where each Y isindividually H or halogen and variables p, q and t are as describedelsewhere herein. In an embodiment, R⁵ and R⁶ are each individuallyhydrogen or a substituted or an unsubstituted C₁-C₆ alkyl. In anembodiment, R⁵ and R⁶ are both hydrogen. In an embodiment, R⁵ and R⁶ areeach individually a substituted or an unsubstituted C₁-C₆ alkyl.

In various embodiments, R⁷ in Formula (II) is H, —COR⁸, —CO₂R⁸,—(CO)—NHR⁸, L⁴, L⁵, L⁶, or L⁷, where each R⁸ is individually asubstituted or an unsubstituted C₁-C₆ alkyl-X³, a substituted or anunsubstituted C₁-C₆ haloalkyl-X³, or —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₂—X³.In an embodiment, R⁷ is H. In an embodiment, R⁷ is —COR⁸. In anembodiment, R⁷ is —CO₂R⁸. In an embodiment, R⁷ is —(CO)—NHR⁸. Thoseskilled in the art will appreciate that when R⁷ is H, —COR⁸, —CO₂R⁸, or—(CO)—NHR⁸, connection of the compound of Formula (II) to the linker-L²-L³-L⁴-L⁵-L⁶-L⁷- is via R⁴ as described elsewhere herein.

In various embodiments, each R⁸ in Formula (II) is individually asubstituted or an unsubstituted C₁-C₆ alkyl-X³, a substituted or anunsubstituted C₁-C₆ haloalkyl-X³, or —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₂—X³,where X³ is —H, —OH, —SH, or —NH₂. In an embodiment, each R⁸ isindividually a substituted or an unsubstituted C₁-C₆ alkyl-X³. In anembodiment, each R⁸ is individually a substituted or an unsubstitutedC₁-C₆ haloalkyl-X³. In an embodiment, each R⁸ is individually—[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₂—X³.

In various embodiments, X² in Formula (II) is —OR⁹, —SR⁹, or —NHR⁹,where R⁹ is H, —COR⁸, —CO₂R⁸, —(CO)—NHR⁸, L⁴, L⁵, L⁶, or L⁷. In anembodiment, X² is —OR⁹. In an embodiment, X² is —SR⁹. In an embodiment,X² is —NHR⁹.

In various embodiments, R⁹ in Formula (II) is H, —COR⁸, —CO₂R⁸,—(CO)—NHR⁸, L⁴, L⁵, L⁶, or L⁷, where R⁸ is a substituted or anunsubstituted C₁-C₆ alkyl-X³, a substituted or an unsubstituted C₁-C₆haloalkyl-X³, or —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₂—X³. In an embodiment, R⁹is H. In an embodiment, R⁹ is —COR⁸. In an embodiment, R⁹ is —CO₂R⁸. Inan embodiment, R⁹ is —(CO)—NHR⁸. Those skilled in the art willappreciate that when R⁹ is H, —COR⁸, —CO₂R⁸, or —(CO)—NHR⁸, connectionof the compound of Formula (II) to the linker -L²-L³-L⁴-L⁵-L⁶-L⁷- is viaR⁷ as described elsewhere herein.

In various embodiments, R⁹ in Formula (II) is L⁴, L⁵, L⁶, or L⁷. In anembodiment, R⁹ is L⁴. In an embodiment, R⁹ is L⁵. In an embodiment, R⁹is L⁶. In an embodiment, R⁹ is L⁷. Those skilled in the art willappreciate that when R⁹ is L⁴, L⁵, L⁶, or L⁷, connection of the compoundof Formula (II) to the linker -L²-L³-L⁴-L⁵-L⁶-L⁷- is via R⁴ as describedelsewhere herein. In an embodiment, exactly one of R⁷ and R⁹ is L⁴, L⁵,L⁶, or L⁷, in which case a single covalent bond links the drug D to thelinker -L²-L³-L⁴-L⁵-L⁶-L⁷- and thereby to Mi.

In various embodiments, each X³ in Formula (II) is individually —H, —OH,—SH, or —NH₂. In an embodiment, X³ is —H. In an embodiment, X³ is —OH.In an embodiment, X³ is —SH. In an embodiment, X³ is —NH₂.

In various embodiments, m in Formula (II) is 1 or 2. In an embodiment, mis 1. In another embodiment, m is 2.

In various embodiments, n⁴ and n⁵ in Formula (II) are each individually0, 1 or 2, with the proviso that n⁴ and n⁵ are not both 0. In anembodiment, n⁴ and n⁵ are both 1. In an embodiment, n⁴ is 0 and n⁵ is 1.In an embodiment, n⁴ is 0 and n⁵ is 2. In an embodiment, n⁴ is 1 and n⁵is 0. In an embodiment, n⁴ is 2 and n⁵ is 0.

In various embodiments, n⁶ in Formula (II) is 0, 1 or 2. In anembodiment, n⁶ is 0, in which case X¹ is —S— or —NH—S—. In anembodiment, n⁶ is 1, in which case X¹ is —S(═O)— or —NH—S(═O)—. In anembodiment, n⁶ is 2, in which case X¹ is —S(═O)₂— or —NH—S(═O)₂—.

In various embodiments, each Y in Formula (II) is individually H orhalogen. In an embodiment, each Y is hydrogen. In an embodiment, —CY₂ isCH₂. In an embodiment, —CY₃ is CH₃. In an embodiment, —CY₃ is CHF₂. Inan embodiment, —CH₂F is CH₃. In an embodiment, —CY₃ is CF₃.

In various embodiments, each p in Formula (II) is individually 1, 2, 3,4, 5, or 6. In an embodiment, p is 1. In an embodiment, p is 2.

In various embodiments, each q in Formula (II) is individually 0, 1, 2,3, 4, 5, or 6. In an embodiment, q is 1. In an embodiment, q is 2.

In various embodiments, each t in Formula (II) is individually 1, 2, 3,4, 5, or 6. In an embodiment, t is 1. In an embodiment, p is t.

In various embodiments, Formula (II) does not represent deruxtecan orexatecan.

Immunoconjugates

Various embodiments disclosed herein relate to an immunoconjugate ofFormula (I), having the structure:

Ab-[S-L¹-L²-L³-L⁴-L⁵-L⁶-L⁷-D]_(n)   (I)

In various embodiments, L¹ in Formula (III) is L¹ is

In various embodiments, L² in Formula (III) is absent,

where Z¹ and Z² are each individually hydrogen, halogen, NO₂, —O—(C₁-C₆alkyl), or C₁-C₆ alkyl. In an embodiment, L² in Formula (III) is absent.In an embodiment, L² in Formula (III) is

In an embodiment, L² in Formula (III) is

In various embodiments, Z¹ and Z² in Formula (III) are each individuallyhydrogen, halogen, NO₂, —O—(C₁-C₆ alkyl), or C₁-C₆ alkyl. In anembodiment, at least one of Z¹ and Z² is hydrogen. In an embodiment, atleast one of Z¹ and Z² is halogen. In an embodiment, at least one of Z¹and Z² is NO₂. In an embodiment, at least one of Z¹ and Z² is —O—(C₁-C₆alkyl). For example, in an embodiment, at least one of Z¹ and Z² ismethoxy. In an embodiment, at least one of Z¹ and Z² is C₁-C₆ alkyl. Forexample, in an embodiment, at least one of Z¹ and Z² is methyl.

In various embodiments, L³ in Formula (III) is —(CH₂)n¹-C(═O)— or—(CH₂CH₂O)n¹-(CH₂)n¹C(═O)—, where n¹ are independently integers of 0 to12. In an embodiment, L³ is —(CH₂)n¹-C(═O)—. For example, in anembodiment, L³ is —C(═O)—. In an embodiment, L³ is—(CH₂CH₂O)n¹-(CH₂)n¹C(═O)—. For example, in an embodiment, L³ is—CH₂C(═O)—. In embodiment, n¹ is an integer of 1 to 12, such as 1 to 6or 1 to 3.

In various embodiments, L⁴ in Formula (III) is a tetrapeptide residue.For example, in an embodiment, L⁴ is a tetrapeptide residue selectedfrom SEQ ID NO: 43 GGFG (gly-gly-phe-gly), SEQ ID NO: 44 EGGF(glu-gly-gly-phe), SEQ ID NO: 45 SGGF (ser-gly-gly-phe), and SEQ ID NO:46 KGGF (lys-gly-gly-phe).

In various embodiments, L⁵ in Formula (III) is absent or—[NH(CH₂)n²]n³-, where n² is an integer of 0 to 6 and n³ is an integerof 0 to 2. In an embodiment, L⁵ is absent. In an embodiment, L⁵ is—[NH(CH₂)n²]_(n) ³—. For example, in an embodiment, L⁵ is —NH—. Inanother embodiment, L⁵ is —NHCH₂—.

In various embodiments, L⁶ in Formula (III) is absent or

In an embodiment, L⁶ is absent. In another embodiment, L⁶ is

In various embodiments, L⁷ in Formula (III) is absent,

In an embodiment, L⁷ is absent.In an embodiment, L⁷ is

In an embodiment, L⁷ is

In an embodiment, L⁷ is

In an embodiment, L⁷ is

In various embodiments, D in the immunoconjugate of Formula (I) is adrug moiety as described herein (e.g., under the heading “Drug Moieties”above). In an embodiment, D is a cytotoxic anti-cancer drug moiety. Inan embodiment, the drug moiety is exatecan.

In various embodiments, Ab in Formula (III) is an antibody or anantigen-binding fragment. In an embodiment, Ab specifically binds tohuman receptor tyrosine kinase like orphan receptor 1 (ROR1). In anembodiment, Ab binds to a cancer cell surface. In an embodiment, Ab isan anti-HER2 antibody.

In various embodiments, the immunoconjugate of Formula (I) isrepresented by a structure selected from the following, for which Z¹ andZ² are each individually selected from hydrogen, fluoro, chloro, —NO₂,and —OCH₃:

Pharmaceutical Compositions

Some embodiments described herein relate to a pharmaceuticalcomposition, that can include an effective amount of one or morecompounds described herein (for example, an immunoconjugate compound ofFormula (I), a drug compound of the Formula (IV), or a pharmaceuticallyacceptable salt thereof) and a pharmaceutically acceptable carrier,diluent, excipient or combination thereof.

The term “pharmaceutical composition” refers to a mixture of one or morecompounds and/or salts disclosed herein with other chemical components,such as diluents or carriers. The pharmaceutical composition facilitatesadministration of the compound to an organism. Pharmaceuticalcompositions can also be obtained by reacting compounds with inorganicor organic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonicacid, p-toluenesulfonic acid and salicylic acid. Pharmaceuticalcompositions will generally be tailored to the specific intended routeof administration.

The term “physiologically acceptable” defines a carrier, diluent orexcipient that does not abrogate the biological activity and propertiesof the compound nor cause appreciable damage or injury to an animal towhich delivery of the composition is intended.

As used herein, a “carrier” refers to a compound that facilitates theincorporation of a compound into cells or tissues. For example, withoutlimitation, dimethyl sulfoxide (DMSO) is a commonly utilized carrierthat facilitates the uptake of many organic compounds into cells ortissues of a subject.

As used herein, a “diluent” refers to an ingredient in a pharmaceuticalcomposition that lacks appreciable pharmacological activity but may bepharmaceutically necessary or desirable. For example, a diluent may beused to increase the bulk of a potent drug whose mass is too small formanufacture and/or administration. It may also be a liquid for thedissolution of a drug to be administered by injection, ingestion orinhalation. A common form of diluent in the art is a buffered aqueoussolution such as, without limitation, phosphate buffered saline thatmimics the pH and isotonicity of human blood.

As used herein, an “excipient” refers to an essentially inert substancethat is added to a pharmaceutical composition to provide, withoutlimitation, bulk, consistency, stability, binding ability, lubrication,disintegrating ability etc., to the composition. For example,stabilizers such as anti-oxidants and metal-chelating agents areexcipients. In an embodiment, the pharmaceutical composition comprisesan anti-oxidant and/or a metal-chelating agent. A “diluent” is a type ofexcipient.

The pharmaceutical compositions described herein can be administered toa human patient per se, or in pharmaceutical compositions where they aremixed with other active ingredients, as in combination therapy, orcarriers, diluents, excipients or combinations thereof. Properformulation is dependent upon the route of administration chosen.Techniques for formulation and administration of the compounds describedherein are known to those skilled in the art.

The pharmaceutical compositions disclosed herein may be manufactured ina manner that is itself known, e.g., by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or tableting processes. Additionally, theactive ingredients are contained in an amount effective to achieve itsintended purpose. Many of the compounds used in the pharmaceuticalcombinations disclosed herein may be provided as salts withpharmaceutically compatible counterions.

Multiple techniques of administering a compound, salt and/or compositionexist in the art including, but not limited to, oral, rectal, pulmonary,topical, aerosol, injection, infusion and parenteral delivery, includingintramuscular, subcutaneous, intravenous, intramedullary injections,intrathecal, direct intraventricular, intraperitoneal, intranasal andintraocular injections. In some embodiments, a compound of Formula (I),or a pharmaceutically acceptable salt thereof, can be administeredorally.

One may also administer the compound, salt and/or composition in a localrather than systemic manner, for example, via injection or implantationof the compound directly into the affected area, often in a depot orsustained release formulation. Furthermore, one may administer thecompound in a targeted drug delivery system, for example, in a liposomecoated with a targeting ligand to a specific cell or tissue type. Theliposomes will be targeted to and taken up selectively by the targetedcell or tissue.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration. The pack or dispensermay also be accompanied with a notice associated with the container inform prescribed by a governmental agency regulating the manufacture,use, or sale of pharmaceuticals, which notice is reflective of approvalby the agency of the form of the drug for human or veterinaryadministration. Such notice, for example, may be the labeling approvedby the U.S. Food and Drug Administration for prescription drugs, or theapproved product insert. Compositions that can include a compound and/orsalt described herein formulated in a compatible pharmaceutical carriermay also be prepared, placed in an appropriate container and labeled fortreatment of an indicated condition.

Uses and Methods of Treatment

Some embodiments described herein relate to a method for treating acancer or a tumor described herein that can include administering aneffective amount of a compound described herein (for example, animmunoconjugate compound of Formula (I), a drug compound of the Formula(IV), or a pharmaceutically acceptable salt thereof) or a pharmaceuticalcomposition that includes a compound described herein (for example, animmunoconjugate compound of Formula (I), a drug compound of the Formula(IV), or a pharmaceutically acceptable salt thereof) to a subject havingthe cancer or tumor. Other embodiments described herein relate to theuse of an effective amount of a compound described herein (for example,an immunoconjugate compound of Formula (I), a drug compound of theFormula (IV), or a pharmaceutically acceptable salt thereof) or apharmaceutical composition that includes a compound described herein(for example, an immunoconjugate compound of Formula (I), a drugcompound of the Formula (IV), or a pharmaceutically acceptable saltthereof) in the manufacture of a medicament for treating a cancer or atumor described herein. Still other embodiments described herein relateto an effective amount of a compound described herein (for example, animmunoconjugate compound of Formula (I), a drug compound of the Formula(IV), or a pharmaceutically acceptable salt thereof) or a pharmaceuticalcomposition that includes a compound described herein (for example, animmunoconjugate compound of Formula (I), a drug compound of the Formula(IV), or a pharmaceutically acceptable salt thereof) for treating acancer or a tumor described herein.

Examples of cancers and tumors include but are not limited to: lungcancer, urothelial cancer, colorectal cancer, prostate cancer, ovariancancer, pancreatic cancer, breast cancer, bladder cancer, gastriccancer, gastrointestinal stromal tumor, uterine cervix cancer,esophageal cancer, squamous cell carcinoma, peritoneal cancer, livercancer, hepatocellular cancer, colon cancer, rectal cancer, colorectalcancer, endometrial cancer, uterine cancer, salivary gland cancer,kidney cancer, vulval cancer, thyroid cancer, penis cancer, leukemia,malignant lymphoma, plasmacytoma, myeloma, or sarcoma.

As used herein, a “subject” refers to an animal that is the object oftreatment, observation or experiment. “Animal” includes cold- andwarm-blooded vertebrates and invertebrates such as fish, shellfish,reptiles and, in particular, mammals. “Mammal” includes, withoutlimitation, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats,cows, horses, primates, such as monkeys, chimpanzees, and apes, and, inparticular, humans. In some embodiments, the subject can be human. Insome embodiments, the subject can be a child and/or an infant, forexample, a child or infant with a fever. In other embodiments, thesubject can be an adult.

As used herein, the terms “treat,” “treating,” “treatment,”“therapeutic,” and “therapy” do not necessarily mean total cure orabolition of the disease or condition. Any alleviation of any undesiredsigns or symptoms of the disease or condition, to any extent can beconsidered treatment and/or therapy. Furthermore, treatment may includeacts that may worsen the subject's overall feeling of well-being orappearance.

The terms “therapeutically effective amount” and “effective amount” areused to indicate an amount of an active compound, or pharmaceuticalagent, that elicits the biological or medicinal response indicated. Forexample, a therapeutically effective amount of compound, salt orcomposition can be the amount needed to prevent, alleviate or amelioratesymptoms of the disease or condition, or prolong the survival of thesubject being treated. This response may occur in a tissue, system,animal or human and includes alleviation of the signs or symptoms of thedisease or condition being treated. Determination of an effective amountis well within the capability of those skilled in the art, in view ofthe disclosure provided herein. The therapeutically effective amount ofthe compounds disclosed herein required as a dose will depend on theroute of administration, the type of animal, including human, beingtreated and the physical characteristics of the specific animal underconsideration. The dose can be tailored to achieve a desired effect, butwill depend on such factors as weight, diet, concurrent medication andother factors which those skilled in the medical arts will recognize.

For example, an effective amount of a compound is the amount thatresults in: (a) the reduction, alleviation or disappearance of one ormore symptoms caused by the cancer, (b) the reduction of tumor size, (c)the elimination of the tumor, and/or (d) long-term disease stabilization(growth arrest) of the tumor. In the treatment of lung cancer (such asnon-small cell lung cancer), a therapeutically effective amount is thatamount that alleviates or eliminates cough, shortness of breath and/orpain.

The amount of the immunoconjugate compound of Formula (I), drug compoundof the Formula (IV), or pharmaceutically acceptable salt thereof,required for use in treatment will vary not only with the particularcompound or salt selected but also with the route of administration, thenature and/or symptoms of the disease or condition being treated and theage and condition of the patient and will be ultimately at thediscretion of the attendant physician or clinician. In cases ofadministration of a pharmaceutically acceptable salt, dosages may becalculated as the free base. As will be understood by those of skill inthe art, in certain situations it may be necessary to administer thecompounds disclosed herein in amounts that exceed, or even far exceed,the dosage ranges described herein in order to effectively andaggressively treat particularly aggressive diseases or conditions.

In general, however, a suitable dose will often be in the range of fromabout 0.05 mg/kg to about 10 mg/kg. For example, a suitable dose may bein the range from about 0.10 mg/kg to about 7.5 mg/kg of body weight perday, such as about 0.15 mg/kg to about 5.0 mg/kg of body weight of therecipient per day, about 0.2 mg/kg to 4.0 mg/kg of body weight of therecipient per day, or any amount in between. The compound may beadministered in unit dosage form; for example, containing 1 to 500 mg,10 to 100 mg, 5 to 50 mg or any amount in between, of active ingredientper unit dosage form.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations.

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight, the severity of theaffliction, the mammalian species treated, the particular compoundsemployed and the specific use for which these compounds are employed.The determination of effective dosage levels, that is the dosage levelsnecessary to achieve the desired result, can be accomplished by oneskilled in the art using routine methods, for example, human clinicaltrials, in vivo studies and in vitro studies. For example, usefuldosages of an immunoconjugate compound of Formula (I), a drug compoundof the Formula (IV), or a pharmaceutically acceptable salt thereof, canbe determined by comparing their in vitro activity and in vivo activityin animal models. Such comparison can be done by comparison against anestablished drug, such as cisplatin and/or gemcitabine.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain themodulating effects, or minimal effective concentration (MEC). The MECwill vary for each compound but can be estimated from in vivo and/or invitro data. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. However, HPLCassays or bioassays can be used to determine plasma concentrations.Dosage intervals can also be determined using MEC value. Compositionsshould be administered using a regimen which maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%. In cases of local administration or selectiveuptake, the effective local concentration of the drug may not be relatedto plasma concentration.

It should be noted that the attending physician would know how to andwhen to terminate, interrupt or adjust administration due to toxicity ororgan dysfunctions. Conversely, the attending physician would also knowto adjust treatment to higher levels if the clinical response were notadequate (precluding toxicity). The magnitude of an administrated dosein the management of the disorder of interest will vary with theseverity of the disease or condition to be treated and to the route ofadministration. The severity of the disease or condition may, forexample, be evaluated, in part, by standard prognostic evaluationmethods. Further, the dose and perhaps dose frequency, will also varyaccording to the age, body weight and response of the individualpatient. A program comparable to that discussed above may be used inveterinary medicine.

Compounds, salts and compositions disclosed herein can be evaluated forefficacy and toxicity using known methods. For example, the toxicologyof a particular compound, or of a subset of the compounds, sharingcertain chemical moieties, may be established by determining in vitrotoxicity towards a cell line, such as a mammalian, and preferably human,cell line. The results of such studies are often predictive of toxicityin animals, such as mammals, or more specifically, humans.Alternatively, the toxicity of particular compounds in an animal model,such as mice, rats, rabbits, dogs or monkeys, may be determined usingknown methods. The efficacy of a particular compound may be establishedusing several recognized methods, such as in vitro methods, animalmodels, or human clinical trials. When selecting a model to determineefficacy, the skilled artisan can be guided by the state of the art tochoose an appropriate model, dose, route of administration and/orregime.

Synthesis

Drug compounds of the Formula (IV), or pharmaceutically acceptable saltsthereof, can be made in various ways by those skilled using knowntechniques as guided by the detailed teachings provided herein. Forexample, in an embodiment, drug compounds of the Formula (IV) areprepared in accordance with the general schemes illustrated in FIGS. 2-4.

Conjugates of the Formula (III) can be made in various ways by thoseskilled using known techniques as guided by the detailed teachingsprovided herein. For example, in an embodiment, conjugates of theFormula (II) are prepared in accordance with the general schemesillustrated in FIGS. 5-10 . Although illustrated with specific linkersand payloads, those skilled in the art will appreciate that otherlinkers and/or payloads may be used in similar manners.

Immunoconjugates of the Formula (I) can be made in various ways by thoseskilled using known techniques as guided by the detailed teachingsprovided herein. For example, in an embodiment, immunoconjugates of theFormula (I) are prepared in accordance with the general schemeillustrated in FIG. 11 . In an embodiment, a process of producing animmunoconjugate as described herein comprises reacting an effectiveamount of a thiol-functionalized antibody or antigen-binding fragmentwith a conjugate as described herein under reaction conditions effectiveto form the immunoconjugate. In an embodiment, the process furthercomprises reducing an antibody or an antigen-binding fragment underreducing conditions effective to form the thiol-functionalized antibodyor antigen-binding fragment.

EXAMPLES

Additional embodiments are disclosed in further detail in the followingexamples, which are not in any way intended to limit the scope of theclaims.

Example 1N-((1S,8S)-8-ethyl-4-fluoro-8-hydroxy-3-methyl-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopenta[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide(1-14) (FIG. 12)

1-Bromo-3-fluoro-2-methyl-5-nitrobenzene (1-2)

A mixture of 2-fluoro-1-methyl-4-nitrobenzene (1-1) (25.0 g, 322 mmol,1.0 equiv) in heptane (250 mL) and H₂SO₄ (250 mL) was heated at 70° C.Then N-bromosuccinimide (68.84 g, 386.78 mmol, 1.2 eq) was addedportion-wise to the above mixture at 70° C. The resulting red suspensionwas stirred at 70° C. for 15 h. TLC (petroleum ether/ethyl acetate=10/1,R_(f)=0.6) showed that a new main spot was formed. The reaction mixturewas poured into ice water (1 L) and extracted with ethyl acetate (3×500mL). The combined organic phases were washed with brine (500 mL), driedover Na₂SO₄, filtered, and concentrated under reduced pressure. Theresidue was purified by column chromatography on silica gel eluting withpetroleum ether to give 1-bromo-3-fluoro-2-methyl-5-nitrobenzene (1-2)(17.0 g, 20% yield). ¹H NMR (400 MHz, CDCl₃) δ ppm 8.27 (t, J=1.79 Hz,1H), 7.89 (dd, J=8.76, 2.21 Hz, 1H), 2.44 (d, J=2.50 Hz, 3H).

3-Bromo-5-fluoro-4-methylaniline (1-3)

To a solution of bromo-3-fluoro-2-methyl-5-nitrobenzene (1-2) (15.0 g,64.1 mmol, 1.0 equiv) in ethanol (750 mL) and water (150 mL) were addediron powder (10.7 g, 192 mmol, 3.0 equiv) and NH₄Cl (6.86 g, 128 mmol,2.0 equiv). The suspension was stirred at 80° C. for 2 h. TLC (petroleumether/ethyl acetate=10/1, R_(f)=0.3) showed that a new main spot wasformed. After cooling to 25° C., the reaction mixture was filteredthrough Celite pad, washing with ethanol (500 mL). The combinedfiltrates were concentrated to dryness and the residue was purified bycolumn chromatography on silica gel eluting with 9% ethyl acetate inpetroleum ether to give 3-bromo-5-fluoro-4-methylaniline (1-3) (8.0 g,55% yield). ¹H NMR (400 MHz, CDCl₃) δ ppm 6.67-6.73 (m, 1H), 6.34 (dd,J=10.97, 2.27 Hz, 1H), 3.65 (br s, 2H), 2.20 (d, J=2.15 Hz, 3H). ¹⁹F NMR(400 MHz, CDCl₃) δ ppm −111.86.

N-(3-Bromo-5-fluoro-4-methylphenyl)acetamide (1-4)

To a mixture of 3-bromo-5-fluoro-4-methylaniline (1-3) (8.00 g, 39.2mmol, 1.0 equiv) in ethyl acetate (100 mL) were added triethylamine(8.13 g, 80.4 mmol, 11.2 mL, 2.05 equiv) and acetic anhydride (5.20 g,51.0 mmol, 4.77 mL, 1.3 equiv). The reaction mixture was stirred at 25°C. for 3 h. TLC (petroleum ether/ethyl acetate=3/1, R_(f)=0.25) showedthat a new main spot was formed. The reaction mixture was quenched withwater and extracted with ethyl acetate (3×50 mL). The combined organicphases were washed with brine (100 mL), dried over Na₂SO₄, filtered, andconcentrated under reduced pressure to giveN-(3-bromo-5-fluoro-4-methyl-phenyl)acetamide (1-4) (8.8 g, 91% yield).¹H NMR (400 MHz, CD₃OD) δ ppm 7.59 (d, J=1.53 Hz, 1H), 7.41 (dd,J=11.55, 2.02 Hz, 1H), 2.26 (d, J=2.20 Hz, 3H), 2.11 (s, 3H).

tert-Butyl (E)-3-(5-acetamido-3-fluoro-2-methylphenyl)acylate (1-5)

To an orange solution of N-(3-bromo-5-fluoro-4-methyl-phenyl)acetamide(1-4) (8.00 g, 32.5 mmol, 1.0 equiv) and tert-butyl acrylate (4.58 g,35.8 mmol, 5.19 mL, 1.1 equiv) in dioxane (100 mL) were addedN-cyclohexyl-N-methylcyclohexanamine (6.99 g, 35.8 mmol, 7.58 mL, 1.1equiv) and Pd(t-Bu₃P)₂ (831 mg, 1.63 mmol, 0.05 equiv). The reactionmixture was stirred at 100° C. for 16 h under nitrogen atmosphere. LCMS(retention time=0.797 min) showed the formation of desired product. Thereaction mixture was quenched with water (100 mL) and extracted withethyl acetate (3×100 mL). The combined organic layers were washed withbrine (200 mL), dried over Na₂SO₄, filtered and concentrated underreduced pressure to dryness. The residue was purified by columnchromatography on silica gel, eluting with 16% of ethyl acetate inpetroleum ether to give tert-butyl(E)-3-(5-acetamido-3-fluoro-2-methylphenyl)acylate (1-5) (10.0 g, 94%yield). ¹H NMR (400 MHz, DMSO-D₆) δ ppm 10.06 (s, 1H), 7.72 (d, J=15.77Hz, 1H), 7.61 (s, 1H), 7.51 (dd, J=11.98, 1.47 Hz, 1H), 6.22 (d, J=15.77Hz, 1H), 2.20 (d, J=1.59 Hz, 3H), 2.04 (s, 3H) 1.49 (s, 9H). ¹⁹F NMR(400 MHz, CDCl₃) δ ppm −115.02. LCMS (ESI+) m/z: [MH]⁺ calcd forC₁₆H₂₁FNO₃ ⁺: 294.1, found: 294.0.

tert-Butyl 3-(5-acetamido-3-fluoro-2-methylphenyl)propanoate (1-6)

To a solution of tert-butyl(E)-3-(5-acetamido-3-fluoro-2-methylphenyl)acrylate (1-5) (2.80 g, 9.55mmol, 1.0 equiv) in dichloromethane (20 mL) and methanol (20 mL) wasadded Pd/C (10 wt % (1.01 g, 0.1 equiv) under argon atmosphere. Thesuspension was degassed under vacuum and purged with H₂ three times. Themixture was stirred under H₂ (15 psi) at 25° C. for 12 h. TLC (petroleumether/ethyl acetate=3/1, R_(f)=0.2) showed that a new major spot wasformed. After the H₂ atmosphere was exchanged with argon, it wasfiltered through a pad of celite and the filter cake was washed withmethanol (100 mL). The combined filtrates were concentrated underreduced pressure to give tert-butyl3-(5-acetamido-3-fluoro-2-methylphenyl)propanoate (1-6) (2.6 g, 83%yield). ¹H NMR (400 MHz, CD₃OD) δ ppm 7.33 (dd, J=11.80, 2.02 Hz, 1H),7.06 (s, 1H), 2.88 (t, J=7.64 Hz, 2H), 2.49 (t, J=7.70 Hz, 2H), 2.17 (d,J=1.96 Hz, 3H), 2.10 (s, 3H), 1.41 (s, 9H).

3-(5-Acetamido-3-fluoro-2-methylphenyl)propanoic acid (1-7)

To a solution of tert-butyl3-(5-acetamido-3-fluoro-2-methylphenyl)propanoate (1-6) (2.60 g, 8.80mmol, 1.0 equiv) in dichloromethane (30 mL) was added trifluoroaceticacid (10 mL) at 25° C. The reaction mixture was stirred at 25° C. for 12h. TLC (petroleum ether/ethyl acetate=3/1, R_(f)=0.01) showed that a newmajor spot was formed. The reaction mixture was concentrated to give3-(5-acetamido-3-fluoro-2-methylphenyl)propanoic acid (1-7) (2.1 g, 89%yield). ¹H NMR (400 MHz, CD₃OD) δ ppm 7.35 (dd, J=11.86, 1.83 Hz, 1H),7.04 (s, 1H), 2.91 (t, J=7.76 Hz, 2H), 2.55 (t, J=7.83 Hz, 2H), 2.17 (d,J=1.83 Hz, 3H), 2.10 (s, 3H). ¹⁹F NMR (400 MHz, CDCl₃) δ ppm −117.33 ,−77.77.

N-(6-Fluoro-7-methyl-3-oxo-2,3-dihydro-1H-inden-4-yl)acetamide (1-8)

To a solution of 3-(5-acetamido-3-fluoro-2-methyl-phenyl)propanoic acid(1-7) (2.10 g, 8.78 mmol, 1.0 equiv) in trifluoroacetic acid (7 mL) wasadded trifluoroacetic anhydride (7.37 g, 35.1 mmol, 4.88 mL, 4.0 equiv).The reaction mixture was stirred at 60° C. for 15 h. LCMS showed theformation of desired product. The reaction mixture was diluted with 50%acetonitrile/H₂O solution (100 mL), and the pH was adjusted to ˜7 byaddition of 25% aqueous NaOH solution at 0° C. The mixture was extractedwith ethyl acetate (3×100 mL). The combined organic layers were washedwith brine (100 mL), dried over Na₂SO₄, filtered and the concentratedunder reduced pressure to giveN-(6-fluoro-7-methyl-3-oxo-2,3-dihydro-1H-inden-4-yl)acetamide (1-8)(1.5 g, 69% yield). ¹H NMR (400 MHz, CD₃OD) δ ppm 8.02 (d, J=12.57 Hz,1H), 3.01-3.08 (m, 2H), 2.72-2.79 (m, 2H), 2.18-2.23 (m, 6H). LCMS(ESI+) m/z: [MH]⁺ calcd for C₁₂H₁₃FNO₂ ⁺: 222.1, found: 222.0.

N-(6-Fluoro-2-hydroxyimino-7-methyl-3-oxo-2,3-dihydro-1H-inden-4-yl)acetamide (1-9)

A solution of potassium tert-butoxide (1.19 g, 10.58 mmol, 1.3 equiv) intetrahydrofuran (12 mL), ethanol (2.4 mL) and butanol (2.4 mL) werestirred at 0° C. for 0.5 h. Isoamyl nitrite (1.43 g, 12.2 mmol, 1.64 mL,1.5 equiv) andN-(6-fluoro-7-methyl-3-oxo-2,3-dihydro-1H-inden-4-yl)acetamide (1-8)(1.80 g, 8.14 mmol, 1.0 equiv) were added to the above mixture. Thereaction mixture was stirred at 20° C. for 3 h. TLC (petroleumether/ethyl acetate=1/1, R_(f)=0.4) showed that a new major spot wasformed. The resulting red suspension was cooled to 0° C. and quenchedwith 1 N hydrochloric acid solution (50 mL) and extracted with ethylacetate (3×50 mL). The combined organic layers were washed with brine(50 mL), dried over Na₂SO₄, filtered and concentrated under reducedpressure to giveN-(6-fluoro-2-hydroxyimino-7-methyl-3-oxo-2,3-dihydro-1H-inden-4-yl)acetamide(1-9) (2.0 g, 88% yield). ¹H NMR (400 MHz, DMSO-D₆) δ ppm 12.81 (s, 1H),10.33 (s, 1H), 8.03 (d, J=12.57 Hz, 1H), 3.71 (s, 2H), 2.20 (s, 3H),2.16 (s, 3H).

N-(2-Amino-6-fluoro-7-methyl-3-oxo-2,3-dihydro-1H-inden-4-yl)acetamidehydrochloric acid salt (1-10)

To a solution ofN-(6-fluoro-2-hydroxyimino-7-methyl-3-oxo-2,3-dihydro-1H-inden-4-yl)acetamide(1-9) (2.00 g, 6.98 mmol, 1 equiv) in methanol (100 mL) were added Pd/C(10 wt %) (1.48 g, 0.2 equiv) and hydrochloric acid (12 M, 1.74 mL, 3equiv) under nitrogen atmosphere. The suspension was degassed undervacuum and purged with H₂ three times. The mixture was stirred under H₂(15 psi) at 20° C. for 3 h. TLC (petroleum ether/ethyl acetate=1/1,R_(f)=0) showed that a new major spot was formed. After the H₂atmosphere was exchanged with nitrogen, it was filtered through a pad ofCelite and washed with methanol (200 mL). The combined filtrates wereconcentrated to dryness to giveN-(2-amino-6-fluoro-7-methyl-3-oxo-2,3-dihydro-1H-inden-4-yl)acetamidehydrochloric acid salt (1-10) (1.5 g, 71% yield). This material was usedin the next step directly without further purification. ¹H NMR (400 MHz,DMSO-D₆) δ ppm 9.99 (s, 1H), 8.02 (d, J=12.59 Hz, 1H), 4.33 (br s, 1H),3.98 (br s, 2H), 3.50 (dd, J=16.99, 8.19 Hz, 1H), 3.00 (dd, J=17.18,4.46 Hz, 1H), 2.21 (s, 3H), 2.18 (s, 3H). ¹⁹F NMR (400 MHz, CDCl₃) δ ppm−102.81.

N-(7-Amino-5-fluoro-4-methyl-1-oxo-2,3-dihydro-1H-inden-2-yl)acetamide(1-11)

To a mixture ofN-(2-amino-6-fluoro-7-methyl-3-oxo-2,3-dihydro-1H-inden-4-yl)acetamidehydrochloric acid salt (1-10) (1.50 g, 6.35 mmol, 1.0 equiv) indichloromethane (45 mL) were added triethylamine (1.93 g, 19.0 mmol,2.65 mL, 3.0 equiv) and acetic anhydride (778 mg, 7.62 mmol, 0.714 mL,1.2 equiv). The reaction mixture was stirred at 25° C. for 2 h. TLC(petroleum ether/ethyl acetate=1/1, R_(f)=0.1) showed that a new majorspot was formed. The reaction mixture was quenched with water (50 mL)and extracted with dichloromethane (3×50 mL). The combined organiclayers were washed with brine (50 mL), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give a red gum.

The gum was dissolved in HCl/MeOH (20 mL, 4 M) and stirred at 25° C. for2 h. LCMS showed formation of the desired product. The resulting redsuspension was concentrated to give a red residue. The residue wasdiluted with methanol (2 mL) and purified by prep-HPLC (column:Phenomenex luna C18 80*40 mm*3 um; mobile phase: [water (HCl)-ACN]; B %:28%-48%, 7 min) to affordN-(7-amino-5-fluoro-4-methyl-1-oxo-2,3-dihydro-1H-inden-2-yl)acetamide(1-11) (0.70 g, 47% yield). ¹H NMR (400 MHz, CD₃OD) δ ppm 6.52 (d,J=11.56 Hz, 1H), 4.48 (dd, J=8.23, 5.01 Hz, 1H), 3.44 (dd, J=17.05, 8.23Hz, 1H), 2.83 (dd, J=17.11, 4.95 Hz, 1H), 2.11 (d, J=1.07 Hz, 3H), 2.02(s, 3H). ¹⁹F NMR (400 MHz, CDCl₃) δ ppm −107.29. ¹³CNMR (100 MHz,CD₃OD): 201.9, 172.1, 167.9, 165.4, 153.8, 153.7, 143.1, 143.0, 117.0,112.6, 112.4, 101.6, 101.3, 55.5, 31.8, 20.8, 8.0. LCMS (ESI+) m/z:[MH]⁺ calcd for C₁₂H₁₄FN₂O₂ ⁺: 237.1, found: 237.0.

(R)—N-(7-Amino-5-fluoro-4-methyl-1-oxo-2,3-dihydro-1H-inden-2-yl]acetamide(1-12-P1) and(S)—N-7-amino-5-fluoro-4-methyl-1-oxo-2,3-didro-1H-inden-2-yl]acetamide(1-12-P2)

N-(7-amino-5-fluoro-4-methyl-1-oxo-2,3-dihydro-1H-inden-2-yl) acetamide(1-11) (0.70 g, 2.96 mmol) was dissolved in MeOH, neutralized with NH₄OHand separated by chiral SFC to afford(R)—N-7-amino-5-fluoro-4-methyl-1-oxo-2,3-dihydro-1H-inden-2-yl]acetamide(1-12-P1) (compound 1-12-P1 may be the opposite enantiomer of thatdepicted) (200 mg, 28% yield) and(S)—N-7-amino-5-fluoro-4-methyl-1-oxo-2,3-dihydro-1H-inden-2-yl]acetamide(1-12-P2) (compound 1-12-P2 may be the opposite enantiomer of thatdepicted) (180 mg, 26% yield). Note: the stereochemistry is arbitrarilyassigned. SFC separation method: column: DAICEL CHIRALPAK AD (250 mm*30mm, 10 um); mobile phase: [0.1% NH₃H₂O IPA]; B %: 30%-30%, 12 min, SFC(1-12-P1, RT=3.012 min) and SFC (1-12-P2, RT=3.270 min).

Spectra for 1-12-P1: ¹H NMR (400 MHz, CD₃OD) δ ppm 2.01 (s, 3H) 2.05 (s,3H) 2.74 (dd, J=16.81, 5.01 Hz, 1H) 3.40 (br d, J=8.70 Hz, 1H) 4.50 (dd,J=8.23, 5.01 Hz, 1H) 6.26 (d, J=12.40 Hz, 1H). ¹⁹F NMR (400 MHz, CDCl₃)δ ppm −108.05. LCMS (ESI+) m/z: [MH]⁺ calcd for C₁₂H₁₄FN₂O₂: 237.10,found: 237.0.

Spectra for 12-P2: ¹H NMR (400 MHz, CD₃OD) δ ppm 2.01 (s, 3H) 2.05 (s,3H) 2.74 (dd, J=17.11, 4.95 Hz, 1H) 3.40 (br d, J=8.82 Hz, 1H) 4.50 (dd,J=8.29, 5.07 Hz, 1H) 6.26 (d, J=12.40 Hz, 1H). ¹⁹F NMR (400 MHz, CDCl₃)δ ppm −108.03. LCMS (ESI+) m/z: [MH]⁺ calcd for C₁₂H₁₄FN₂O₂: 237.10,found: 237.1.

N-((1S,8S)-8-ethyl-4-fluoro-8-hydroxy-3-methyl-9,12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopenta[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide(1-14)

The mixture ofN-(7-amino-5-fluoro-4-methyl-1-oxo-2,3-dihydro-1H-inden-2-yl)acetamide(1-11) (50 mg, 0.21 mmol),(S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (111.4 mg, 0.42 mmol), TsOH (36.2 mg, 0.21 mmol) inxylene (2 mL) was heated at 140° C. for 12 h. All volatiles were removedunder high vacuum, and the residue subjected to chromatography first,then chiral SFC separation to afford 1-14a and 1-14b. LCMS (ESI+) m/z:[MH]⁺ calcd for C₂₅H₂₃FN₃O₅: 464.2, found: 464.3.

Example 2N-((1S,9S)-9-ethyl-9-hydroxy-10,13-dioxo-1,2,9,10,13,15-hexahydro-12H-cyclo-penta[de][1,3]dioxolo[4,5-g]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide(2-30) (FIG. 13)

2-Bromo-6-methoxy-4-nitrophenol (2-16)

To a solution of 2-methoxy-4-nitrophenol (2-15) (50.0 g, 296 mmol, 1.0equiv) in glacial acetic acid (500 mL) was added bromine (52.0 g, 325mmol, 16.8 mL, 1.1 equiv) slowly using a dropping funnel at 20° C. Theresulting mixture was stirred at 20° C. for 2 h. TLC (petroleumether:ethyl acetate=2:1) indicated the starting material was consumedand a major spot with lower polarity was formed. The reaction mixturewas slowly poured into water (1.5 L) while stirring, and the resultingmixture was stirred at 20° C. for additional 10 min. The mixture wasfiltered, and concentrated under reduced pressure to dryness. Theresidue was triturated with water (2×500 mL), the resulting product wascollected by filtration and dried in a vacuum oven to afford2-bromo-6-methoxy-4-nitrophenol (2-16) (60.0 g, 73 yield). ¹H NMR (400MHz, CDCl₃): δ ppm 8.13 (d, J=2.4 Hz, 1H), 7.74 (d, J=2.4 Hz, 1H), 6.61(s, 1H), 4.03 (s, 3H). LCMS (ESI−) m/z: [MH]⁻ calcd for C₇H₅BrNO₄ ⁻:245.9, found: 245.9.

3-Bromo-5-nitrobenzene-1,2-diol (2-17)

To a solution of 2-bromo-6-methoxy-4-nitrophenol (2-16) (30.0 g, 121mmol, 1 eq) in CH₂Cl₂ (1.5 L) was added boron tribromide (45.4 g, 181mmol, 17.5 mL, 1.5 equiv) at 0° C. The reaction mixture was allowed towarm to 30° C. and stirred for 15 h. TLC (petroleum ether:ethylacetate=1:1) indicated the starting material was consumed and a newmajor spot with higher polarity was formed. The reaction mixture wasquenched with methanol (200 mL) and concentrated under reduced pressureto afford 3-bromo-5-nitrobenzene-1,2-diol (2-17) (26.9 g, 95% yield). ¹HNMR (400 MHz, DMSO-D₆): δ ppm 10.94 (s, 2H), 7.87 (d, J=2.8 Hz, 1H),7.62 (d, J=2.8 Hz, 1H).

4-Bromo-6-nitro-1,3-benzodioxole (2-18)

To a solution of 3-bromo-5-nitrobenzene-1,2-diol (2-17) (30.0 g, 128mmol, 1.0 equiv) in N,N-dimethylformamide (1 L) was added Cs₂CO₃ (125 g,385 mmol, 3 equiv) and diiodomethane (54.9 g, 205 mmol, 16.6 mL, 1.6equiv) at 20° C. The reaction mixture was stirred at 100° C. for 12 h.TLC (petroleum ether:ethyl acetate=3:1) indicated the starting materialwas consumed and a new major spot with lower polarity was formed. Thereaction mixture was cooled to 20° C., poured into ice-water (1.5 L),and extracted with ethyl acetate (2×1.5 L). The combined organic layerswere washed with brine (2×1 L), dried over anhydrous Na₂SO₄, filteredand concentrated under reduced pressure to give a brown oil. The oil wastriturated with toluene (20 mL), and the resulting product was collectedby filtration, dried in a vacuum oven to afford4-bromo-6-nitro-1,3-benzodioxole (2-18) (17 g, 48% yield). ¹H NMR (400MHz, CDCl₃): δ ppm 8.06 (d, J=2.0 Hz, 1H), 7.63 (d, J=2.4 Hz, 1H), 6.23(s, 2H).

7-Bromobenzo[d][1,3]dioxol-5-amine (2-19)

To a solution of 4-bromo-6-nitro-1,3-benzodioxole (2-18) (10.0 g, 40.6mmol, 1.0 equiv) in ethanol (500 mL) and water (100 mL) was added iron(6.81 g, 122 mmol, 3.0 equiv) and NH₄Cl (4.35 g, 81.3 mmol, 2.0 equiv)at 20° C. The mixture was stirred at 80° C. for 2 h. TLC (petroleumether:ethyl acetate=1:1) indicated the starting material was consumedand a new major spot with lower polarity was formed. After the reactionmixture was cooled to 20° C., it was filtered through a pad of Celite,washed with ethanol (500 mL). The filtrate was concentrated underreduced pressure and crushed ice was added. The resulting product wascollected by filtration, washed with water, and dried in a vacuum ovento afford 7-bromobenzo[d][1,3]dioxol-5-amine (2-19) (7.5 g, 76% yield).¹H NMR (400 MHz, CDCl₃): δ ppm 6.29 (s, 1H), 6.21 (s, 1H), 5.94 (s, 2H),3.51 (s, 2H).

N-(7-Bromobenzo[d][1,3]dioxol-5-yl)acetamide (2-20)

To a solution of 7-bromo-1,3-benzodioxol-5-amine (2-19) (6.00 g, 27.78mmol, 1.0 equiv) in ethyl acetate (50 mL) was added acetic anhydride(3.40 g, 33.33 mmol, 3.12 mL, 1.2 equiv) and triethylamine (8.43 g, 83.3mmol, 11.6 mL, 3.0 equiv) at 20° C. and the resulting mixture wasstirred for 10 h. TLC (ethyl acetate:methanol=4:1) indicated thestarting material was consumed and a new major spot with higher polaritywas formed. The reaction mixture was quenched by the addition ofsaturated NaHCO₃ (50 mL) at 0° C. and extracted with ethyl acetate (2×50mL). The combined organic layers were dried over Na₂SO₄, filtered, andconcentrated under reduced pressure. The residue was purified by columnchromatography (eluting with petroleum ether/ethyl acetate=1/3 to 1/4)to afford N-(7-bromobenzo[d][1,3]dioxol-5-yl)acetamide (2-20) (5.5 g,69% yield). ¹H NMR (400 MHz, CDCl₃): δ ppm 7.19 (s, 1H), 7.13 (d, J=1.6Hz, 1H), 7.02 (d, J=2.0 Hz, 1H), 6.02 (s, 2H), 2.15 (s, 3H). LCMS (ESI+)m/z: [MH]⁺ calcd for C₉H₉BrNO₃ ⁺: 258.0, found: 257.9.

tert-Butyl (E)-3-(6-acetamido-1,3-benzodioxol-4-yl)acrylate (2-21)

To a solution of N-(7-bromo-1,3-benzodioxol-5-yl)acetamide (2-20) (2.00g, 7.75 mmol, 1.0 equiv) and tert-butyl acrylate (1.09 g, 8.52 mmol,1.24 mL, 1.1 equiv) in dioxane (20 mL) were addedN-cyclohexyl-N-methyl-cyclohexanamine (1.67 g, 8.52 mmol, 1.81 mL, 1.1equiv) and bis(tri-tert-butylphosphine)palladium(0) (198 mg, 0.387 mmol,0.05 equiv) under nitrogen atmosphere. The mixture was stirred at 100°C. for 5 h. TLC (petroleum ether:ethyl acetate=2:1, R_(f)-0.4) and LCMSshowed the starting material was consumed and the desired product wasformed. After cooling down to RT, the reaction mixture was filtered, andthe filtrate was concentrated to give a product that was then trituratedwith ethyl acetate (40 mL) at 20° C. for 10 min, then filtered and thefiltrate was concentrated under reduced pressure to give tert-butyl(E)-3-(6-acetamido-1,3-benzodioxol-4-yl)acrylate (2-21) (2.0 g, 76%yield). ¹H NMR (400 MHz, CDCl₃) δ ppm 7.44 (d, J=16.09 Hz, 1H) 7.23 (d,J=1.91 Hz, 1H) 7.05 (br s, 1H) 6.86 (d, J=1.91 Hz, 1H) 6.55 (d, J=15.97Hz, 1H) 6.06 (s, 2H) 2.17 (s, 3H) 1.53 (s, 9H). LCMS (ESI+) m/z: [M−56]⁺calcd for C₁₆H₂₀NO₅—C₄H₉ (^(t)Bu)+H]⁺: 249.1, found: 249.9.

tert-Butyl 3-(6-acetamido-1,3-benzodioxol-4-yl)propanoate (2-22)

To a suspension of Pd/C (2.90 g, 2.46 mmol, 10% purity, 0.5 equiv) inMeOH (20 mL) was added a solution of tert-butyl(E)-3-(6-acetamido-1,3-benzodioxol-4-yl)acrylate (2-21) (1.50 g, 4.91mmol, 1.0 equiv) in MeOH (20 mL) at 20° C. The mixture was hydrogenatedat 20° C. for 2 h under 15 psi of hydrogen atmosphere. TLC (petroleumether/ethyl acetate=1/1, R_(f)-0.48) showed the starting material wasconsumed and a new spot was formed. After the H₂ atmosphere wasexchanged with nitrogen, the reaction mixture was filtered, and filtratewas concentrated to give tert-butyl 3-(6-acetamido-1,3-benzodioxol-4-yl)propanoate (2-22) (1.4 g, 83% yield). It was used for the next stepwithout further purification. ¹H NMR (400 MHz, CDCl₃) δ ppm 7.09 (d,J=1.96 Hz, 1H), 6.98 (br s, 1H), 6.64 (s, 1H), 5.94 (s, 2H), 2.83 (t,J=7.76 Hz, 2H), 2.51-2.59 (m, 2H), 2.14 (s, 3H), 1.43 (s, 9H). LCMS(ESI+) m/z: [M+Na]⁺ calcd for C₁₆H₂₁NO₅Na⁺: 330.1, found: 330.1.

3-(6-Acetamido-1,3-benzodioxol-4-yl)propanoic acid (2-23)

To a solution of tert-butyl3-(6-acetamido-1,3-benzodioxol-4-yl)propanoate (2-22) (1.40 g, 4.56mmol, 1.0 equiv) in CH₂Cl₂ (15 mL) was added TFA (3 mL) at 20° C. andthe mixture was stirred for 2 h. TLC (petroleum ether:ethyl acetate=0:1,R_(f)=0.48) showed the starting material was consumed and a new spot wasformed. The reaction mixture was concentrated under reduced pressure,and the residue was purified by column chromatography (eluting withpetroleum ether/ethyl acetate=I/O to 1/9) to give3-(6-acetamido-1,3-benzodioxol-4-yl)propanoic acid (2-23) (0.85 g, 71%yield). ¹H NMR (400 MHz, CD₃OD) δ ppm 7.08 (d, J=1.91 Hz, 1H), 6.76 (d,J=1.79 Hz, 1H), 5.93 (s, 2H), 2.80-2.88 (m, 2H), 2.57-2.65 (m, 2H), 2.07(s, 3H). LCMS (ESI+) m/z: [MH]⁺ calcd for C₁₂H₁₄NO₅ ⁺: 252.1, found:251.9.

N-(6-Oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-5-yl) acetamide (2-24)

To a solution of 3-(6-acetamido-1,3-benzodioxol-4-yl)propanoic acid(2-23) (16.0 g, 63.7 mmol, 1.0 equiv) in TFA (64 mL) was added TFAA(53.5 g, 255 mmol, 35.4 mL, 4 equiv) dropwise at 20° C. The resultingsolution was heated to 60° C. for 10 h. TLC (petroleum ether:ethylacetate=2:1, R_(f)=0.40) and LCMS indicated the reaction was completed.After cooling down to RT, the reaction mixture was poured into asolution of acetonitrile (100 mL) and water (100 mL). After cooling to0° C., the pH of the mixture was adjusted to 7 with 25% aqueous sodiumhydroxide (150 mL), and water (100 mL) was added. The resulting mixturewas extracted with ethyl acetate (500 mL×3). The combined organic layerswere washed with brine (800 mL), dried over anhydrous Na₂SO₄, filteredand concentrated under reduced pressure. The residue was triturated withethyl acetate (100 mL), filtered and the filter cake was collected togive N-(6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-5-yl)acetamide(2-24) (10.4 g, 59% yield). ¹H NMR (400 MHz, DMSO-D₆) δ ppm 10.47 (s,1H), 7.85 (s, 1H), 6.15 (s, 2H), 2.84-3.04 (m, 2H), 2.60-2.74 (m, 2H),2.17 (s, 3H). LCMS (ESI+) m/z: [MH]⁺ calcd for C₁₂H₁₂NO_(4+: 234.1),found: 234.0.

N-(7-Hydroxyimino-6-oxo-8H-cyclopenta[g][1,3]benzodioxol-5-yl)acetamide(2-25)

A solution of potassium tert-butoxide (1.0 M, 45.0 mL, 2.1 equiv) in THF(41.6 mL), EtOH (6.7 mL) and n-BuOH (6.7 mL) was stirred at 0° C. for 30min. Isopentyl nitrite (3.01 g, 25.7 mmol, 3.46 mL, 1.2 equiv) andN-(6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-5-yl)acetamide (2-24)(5.00 g, 21.4 mmol, 1.0 equiv) were added to the above solution at 0° C.The resulting mixture was stirred at 20° C. for 3 h. LCMS showed theformation of the desired product. The reaction mixture was quenched by1.0 N hydrochloric acid (200 mL), and ethyl acetate (200 mL) was added.The resulting precipitation was collected by filtration. The filtratewas extracted with ethyl acetate (3×500 mL), the combined organic layerswere washed with brine, dried over sodium sulfate, filtered andconcentrated under reduced pressure. The resulting product was combinedwith the filter cake to giveN-(7-hydroxyimino-6-oxo-8H-cyclopenta[g][1,3]benzodioxol-5-yl)acetamide(2-25) (5.6 g, 99% yield). ¹H NMR (400 MHz, DMSO-D₆) δ ppm 12.71 (s,1H), 10.53 (s, 1 H), 7.89 (s, 1H), 6.19 (s, 2H), 3.63 (s, 2H), 2.17 (s,3H). LCMS (ESI+) m/z: [MH]⁺ calcd for C₁₂H₁N₂O₅ ⁺: 263.1, found: 263.0.

N-(7-Amino-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-5-yl)acetamidehydrochloride (2-26)

To a mixture ofN-(7-hydroxyimino-6-oxo-8H-cyclopenta[g][1,3]benzodioxol-5-yl)acetamide(2-25) (550 mg, 2.10 mmol, 1.0 equiv) and HCl (12 M, 0.26 mL, 1.5 equiv)in MeOH (50 mL) was added Pd/C (10 wt. %) (300 mg). The reaction mixturewas purged with hydrogen gas three times, and stirred at 20° C. for 2 hunder 15 psi of hydrogen atmosphere. LCMS indicated the reaction wascompleted. After the H₂ atmosphere was exchanged with nitrogen, thereaction mixture was filtered through a pad of Celite, and the filtratewas concentrated under reduced pressure to giveN-(7-amino-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-5-yl)acetamidehydrochloride (2-26) (500 mg, 83% yield). It was used in the next stepwithout further purification. ¹H NMR (400 MHz, DMSO-D₆) δ ppm 10.09 (brs, 1H), 9.05 (br s, 2H), 8.82 (br s, 2H), 7.85 (s, 1H), 6.23-6.70 (m,2H), 3.98-4.39 (m, 2H), 3.35-3.48 (m, 1H), 2.16 (s, 3H). LCMS (ESI+)m/z: [MH]⁺ calcd for C₁₂H₁₃N₂O₄ ⁺: 249.1, found: 249.0.

N-(7-Acetamido-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-5-yl)acetamide(2-27)

To a solution ofN-(7-amino-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-5-yl)acetamidehydrochloride (2-26) (4.50 g, 15.8 mmol, 1.0 equiv) in DCM (150 mL) wasadded TEA (4.80 g, 47.4 mmol, 6.60 mL, 3.0 equiv), acetic anhydride(1.94 g, 19.0 mmol, 1.78 mL, 1.2 equiv) at 20° C. and the mixture wasstirred for 3 h. TLC (ethyl acetate, R_(f)=0.40) and LCMS indicated thereaction was completed. The mixture was diluted with DCM (500 mL) andwashed with water (100 mL). The organic layer was dried over anhydrousNa₂SO₄, filtered and concentrated under reduced pressure. The residuewas purified by column chromatography (eluting with petroleumether/ethyl acetate=2/1 to 0/1, 5% THF) to giveN-(7-acetamido-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-5-yl)acetamide(2-27) (2.55 g, 50% yield). ¹H NMR (400 MHz, DMSO-D₆) δ ppm 10.35 (s,1H), 8.53 (d, J=7.51 Hz, 1H), 7.87 (s, 1H), 6.13-6.19 (m, 2H), 4.30-4.37(m, 1H), 3.17-3.32 (m, 1H), 2.79 (dd, J=16.75, 5.07 Hz, 1H), 2.14 (s,3H), 1.85 (s, 3H). LCMS (ESI+) m/z: [MH]⁺ calcd for C₁₄H₁₅N₂O₅ ⁺: 291.1,found: 291.0.

N-(5-Amino-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-7-yl)acetamide(2-28)

To a solution ofN-(7-acetamido-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-5-yl)acetamide (2-27) (2.55 g, 8.78 mmol, 1.0 equiv) in MeOH (110 mL) wasadded HCl/MeOH (4 M, 110 mL, 50 equiv) at 20° C. and the solution wasstirred for 3 h. LCMS indicated the reaction was completed. The reactionmixture was concentrated under reduced pressure, the residue wasdissolved in MeOH (10 mL) and pH was adjusted to 7 with saturatedNaHCO₃, and then extracted with ethyl acetate (20 mL×3). The combinedorganic phases were dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to giveN-(5-amino-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-7-yl)acetamide(2-28) (2.0 g, 91% yield). ¹H NMR (400 MHz, DMSO-D₆) δ ppm 8.40 (d,J=7.89 Hz, 1H), 6.21 (s, 1H), 5.97 (d, J=15.79 Hz, 2H), 4.35 (td,J=8.17, 5.37 Hz, 1H), 3.20 (dt, J=16.55, 8.17 Hz, 1H), 2.62 (dd,J=16.66, 5.26 Hz, 1H), 1.84 (s, 3H). ¹³CNMR (100 MHz, DMSO-D₆): δ 199.6,169.1, 154.3, 145.6, 133.4, 127.7, 111.3, 101.3, 93.5, 54.5, 28.7, 22.3.LCMS (ESI+) m/z: [MH]⁺ calcd for C₁₂H₁₃N₂O₄ ⁺: 249.1, found: 249.0.

N-[(7R)-5-Amino-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-7-yl]acetamide(2-29-P1) andN-[(7S)-5-amino-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-7-yl]acetamide(2-29-P2)

N-(5-Amino-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-7-yl)acetamide(2-28) (1.0 g) was dissolved in MeOH and separated by chiral SFC toaffordN-[(7R)-5-amino-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-7-yl]acetamide(2-29-P1) (compound 22-9-P1 may be the opposite enantiomer of thatdepicted) (250 mg, 24% yield), andN-[(7S)-5-amino-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-7-yl]acetamide(2-29-P2) (compound 2-29-P2 may be the opposite enantiomer of thatdepicted) (250 mg, 24% yield).

SFC separation method: column (column: Phenomenex-Cellulose-2 (250 mm×30mm, 10 um); mobile phase: [Neu-ETOH]; B %: 40%-40%, 5 min). Compounds(2-29-P1, RT=2.719 min) and (2-29-P2, RT=2.942 min) were separated bychiral SFC. Note: The stereochemistry is arbitrarily assigned for2-29-P1 and 2-29-P2.

Spectra for 2-29-P1: ¹H NMR (400 MHz, DMSO-D₆) δ ppm 8.30 (br d, J=7.95Hz, 1H), 6.59 (s, 2H), 6.17 (s, 1H), 5.96 (d, J=15.89 Hz, 2H), 4.36 (td,J=8.16, 5.32 Hz, 1H), 3.16-3.25 (m, 1H), 2.61 (dd, J=16.69, 5.20 Hz,1H), 1.84 (s, 3H). LCMS (ESI+) m/z: [MH]⁺ calcd for C₁₂H₁₃N₂O₄ ⁺: 249.1,found: 248.9.

Spectra for 2-29-P2: ¹H NMR (400 MHz, DMSO-D₆) δ ppm 8.30 (br d, J=7.95Hz, 1H), 6.59 (s, 2H), 6.17 (s, 1H), 5.96 (d, J=15.89 Hz, 2H), 4.36 (td,J=8.19, 5.26 Hz, 1H), 3.21 (dd, J=16.69, 8.50 Hz, 1H), 2.61 (dd,J=16.69, 5.20 Hz, 1H), 1.85 (s, 3H). LCMS (ESI+) m/z: [MH]⁺ calcd forC₁₂H₁₃N₂O₄ ⁺: 249.1, found: 248.9.

The mixture ofN-[(7R)-5-amino-6-oxo-7,8-dihydrocyclopenta[g][1,3]benzodioxol-7-yl]acetamide(2-29-P1) (30 mg, 0.12 mmol),(S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (63.6 mg, 0.24 mmol), TsOH (20.7 mg, 0.12 mmol) inxylene (2 mL) was heated at 140° C. for 12 h. All volatiles were removedunder high vacuum, and the residue subjected to chromatography to afford2-30. LCMS (ESI+) m/z: [MH]⁺ calcd for C₂₅H₂₂N₃O₇: 476.5, found: 476.4.

Example 3(1R,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-34) and(1S,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-35) (FIG. 14)

(S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3-dihydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-1,10,13(9H,12H,15H)-trione (3-32)

To a solution of(1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dionemethanesulfonate (100 mg, 0.230 mmol, 1 equiv) in methanol (20 mL) wasadded basic resin (300 mg) at 25° C. and the mixture was stirred for 30min under ultrasonic wave. The mixture was filtered, and the filtratewas concentrated to give a free base.

To a solution of the above obtained free base in methanol (2 mL) wasadded 3,5-ditert-butyl-1,2-benzoquinone (101 mg, 0.459 mmol, 2 equiv).The reaction mixture was stirred at 60° C. for 10 min. The color of thereaction mixture turned from red to dark yellow. The reaction mixturewas stirred at 25° C. for additional 3 h. TLC (ethylacetate:methanol=8:1) indicated about 10% of unreacted starting materialwas remaining and a new spot was formed. The reaction mixture wasquenched by the addition of oxalic acid (2 M in tetrahydrofuran/H₂O=3:1solution, 0.5 mL) at 25° C. and the mixture was stirred for 3 h. Thereaction mixture was diluted with tetrahydrofuran (5 mL), filtered andthe filtrate was concentrated under reduced pressure. The residue waspurified by preparative-HPLC (under HCl condition) to give(S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3-dihydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-1,10,13(9H,12H,15H)-trione (3-32) (70 mg, 25% yield). ¹H NMR (400 MHz, DMSO-D₆)δ ppm 7.94 (d, J=10.6 Hz, 1H), 7.35 (s, 1H), 5.44 (s, 2H), 5.39 (s, 2H),3.55-3.47 (m, 2H), 3.09 (t, J=7.0 Hz, 2H), 2.46 (s, 3H), 1.92-1.79 (m,2H), 0.91-0.85 (m, 3H). LCMS (ESI) m/z: [M+H⁺] calcd for C₂₄H₂OFN₂O₅ ⁺:435.1, found: 435.2.

Preparative-HPLC Method:

-   -   Instrument: Gilson 281 semi-preparative HPLC system    -   Mobile phase: A: HCl/H₂O=0.040% v/v; B: CH₃CN    -   Column: Phenomenex Luna 80*30 mm*3 um    -   Flow rate: 25 mL/min    -   Monitor wavelength: 220&254 nm

Time B % 0.0 30 8.0 60 8.1 60 8.2 100 10.2 100 10.3 30 11.5 30

(9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano-[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-33)

To a solution of give(S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3-dihydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-1,10,13(9H,12H,15H)-trione (3-32) (70 mg, 0.16 mmol, 1.0 equiv) in methanol (2mL) was added AcOH (27.6 mg, 0.460 mmol, 26.3 μL, 10 equiv) and NaBH₃CN(20.1 mg, 0.322 mmol, 2.0 equiv). The mixture was stirred at 0° C. for10 min, and then at 20° C. for 12 h. TLC (ethyl acetate:methanol=8:1)indicated the starting material was consumed, and a new major spot wasformed. The reaction mixture was quenched by H₂O (0.5 mL) at 25° C. andconcentrated under reduced pressure. The residue was purified bypreparative-HPLC to give(9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (33) (20 mg, 29% yield).

The chiral SFC separation of compound 3-33 afforded(1R,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione(3-34) (compound 3-34 may be the opposite diastereomer of that depicted)(2.9 mg, 4% yield) (Peak 1 in SFC at 1.429 min) and(1S,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione(3-35) (compound 3-35 may be the opposite diastereomer of that depicted)(4.7 mg, 7% yield) (Peak 2 in SFC at 1.534 min). Note: Thestereochemistry is arbitrarily assigned.

Spectra of(1R,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione(3-34): ¹H NMR (400 MHz, DMSO-D₆) δ ppm 7.73 (dd, J=10.8, 1.8 Hz, 7.31(s, 1H), 6.50 (s, 1H), 5.97 (d, J=16.0 Hz, 1H), 5.42 (s, 2H), 5.41 (d,J=19.2 Hz, 1H), 5.28 (d, J=18.6 Hz, 1H), 5.12-5.17 (m, 1H), 3.20-3.26(m, 1H), 2.98-3.05 (m, 1H), 2.31-2.35 (m, 1H), 2.33 (s, 3H), 1.95-2.00(m, 1H), 1.84-1.90 (m, 2H), 0.88 (t, J=7.2 Hz, 3H). ¹⁹F NMR (400 MHz,DMSO-D₆): δ ppm −111.8 (s, 1F). LCMS (ESI) m/z: [MH⁺] calcd forC₂₄H₂₂FN₂O₅ ⁺: 437.1, found: 437.1.

Spectra of(1S,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione(3-35): ¹H NMR (400 MHz, DMSO-D₆) δ ppm 7.73 (dd, J=10.8, 1.8 Hz, 7.31(s, 1H), 6.50 (s, 1H), 5.97 (d, J=16.0 Hz, 1H), 5.42 (s, 2H), 5.41 (d,J=19.2 Hz, 1H), 5.28 (d, J=18.6 Hz, 1H), 5.12-5.17 (m, 1H), 3.20-3.26(m, 1H), 2.98-3.05 (m, 1H), 2.31-2.35 (m, 1H), 2.33 (s, 3H), 1.95-2.00(m, 1H), 1.84-1.90 (m, 2H), 0.88 (t, J=7.2 Hz, 3H). ¹⁹F NMR (400 MHz,DMSO-D₆): δ ppm −111.8 (s, 1F). LCMS (ESI) m/z: [MH⁺] calcd forC₂₄H₂₂FN₂O₅ ⁺: 437.1, found: 437.1.

Preparative-HPLC Method:

-   -   Instrument: Gilson 281 semi-preparative HPLC system    -   Mobile phase: A: HCl/H₂O=0.040% v/v; B: CH₃CN    -   Column: Phenomenex Luna 80*30 mm*3 um    -   Flow rate: 25 mL/min    -   Monitor wavelength: 220 &254 nm

Time B % 0.0 20 8.0 50 8.1 50 8.2 100 10.2 100 10.3 20 11.5 20

SFC Separation Method:

-   -   Instrument: Waters SFC150AP preparative SFC    -   Column: DAICEL CHIRALCEL OD (250 mm*30 mm, 10 um)    -   Mobile phase: A for CO₂ and B for EtOH    -   Gradient: B %=45% isocratic elution mode    -   Flow rate: 70 g/min    -   Wavelength: 220 nm    -   Column temperature: 35 degrees centigrade    -   System back pressure: 120 bar

Example 4(1R,8S)-1-amino-8-ethyl-4-fluoro-8-hydroxy-3-methyl-11,14-dihydro-1H-cyclopenta[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-9,12(2H,8H)-dione hydrochloride (4-36) and(1S,8S)-1-amino-8-ethyl-4-fluoro-8-hydroxy-3-methyl-11,14-dihydro-1H-cyclopenta[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-9,12(2H,8H)-dione hydrochloride (4-37) (FIG. 15)

N-((8S)-8-Ethyl-4-fluoro-8-hydroxy-3-methyl-9,12-dioxo-2,8,9,11,12,14-hexahydro-1H-cyclopenta[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide(1-14)

To a mixture ofN-(7-amino-5-fluoro-4-methyl-1-oxo-2,3-dihydro-1H-inden-2-yl)acetamide(100 mg, 0.423 mmol, 1.0 equiv) (1-11) and(S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (223 mg, 0.847 mmol, 2.0 equiv) in xylene (10 mL) at140° C., was added 4-methylbenzenesulfonic acid (29.1 mg, 0.169 mmol,0.4 equiv), and the mixture was stirred at 140° C. for 36 h in a 40 mLseal tube. It was cooled to room temperature and concentrated underreduced pressure, and the residue purified by column chromatography onsilica gel eluting with 9% of MeOH in dichloromethane to giveN-((8S)-8-ethyl-4-fluoro-8-hydroxy-3-methyl-9,12-dioxo-2,8,9,11,12,14-hexahydro-1H-cyclopenta[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide(1-14) (8.50 mg, 4.3% yield). ¹H NMR (400 MHz, DMSO-D₆) δ ppm 8.65 (d,J=8.11 Hz, 1H), 7.72 (d, J=11.56 Hz, 1H), 7.34 (s, 1H), 6.51 (d, J=2.27Hz, 1H), 5.95 (br d, J=5.01 Hz, 1H), 5.43 (s, 2H), 5.09-5.13 (m, 2H),3.82-3.93 (m, 1H), 3.30 (br s, 1H), 2.38 (s, 3H), 1.94 (d, J=2.86 Hz,3H), 1.87 (br d, J=7.39 Hz, 2H), 0.87 (br d, J=4.41 Hz, 3H). LCMS (ESI+)m/z: [MH]⁺ calcd for C₂₅H₂₃FN₃O₅: 464.1, found: 464.2.

(1R,8S)-1-amino-8-ethyl-4-fluoro-8-hydroxy-3-methyl-11,14-dihydro-1H-cyclopenta[de]-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-9,12(2H,8H)-dione hydrochloride (4-36) and(1S,8S)-1-amino-8-ethyl-4-fluoro-8-hydroxy-3-methyl-11,14-dihydro-1H-cyclo-penta[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-9,12(2H,8H)-dione hydrochloride (4-37)

A solution ofN-((8S)-8-ethyl-4-fluoro-8-hydroxy-3-methyl-9,12-dioxo-2,8,9,11,12,14-hexahydro-1H-cyclopenta[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide(1-14) (150 mg, 0.323 mmol, 1.0 equiv) in 6 N aqueous HCl solution (15mL) was stirred at 60° C. for 15 h in a sealed tube. After cooled downto 25° C., the reaction mixture was concentrated under reduced pressure,the residue diluted with methanol (5 mL) and purified by prep-HPLC togive(1R,8S)-1-amino-8-ethyl-4-fluoro-8-hydroxy-3-methyl-11,14-dihydro-1H-cyclopenta[de]-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-9,12(2H,8H)-dione hydrochloride (4-36) (5.0 mg, 3.3% yield) (compound 4-36may be the opposite enantiomer of that depicted) and(1S,8S)-1-amino-8-ethyl-4-fluoro-8-hydroxy-3-methyl-11,14-dihydro-1H-cyclopenta[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-9,12(2H,8H)-dione hydrochloride (4-37) (3.0 mg, 1.4% yield) (compound 4-37may be the opposite enantiomer of that depicted). Note: thestereochemistry is arbitrarily assigned.

Prep-HPLC Method:

-   -   Instrument: Gilson 281 semi-preparative HPLC system    -   Mobile phase: A: HCl/H₂O=0.040% v/v; B: CH₃CN    -   Column: Phenomenex luna C18 80*40 mm*3 um    -   Flow rate: 40 mL/min    -   Monitor wavelength: 220&254 nm

Time B % 0.0 13 7.0 38 7.1 38 7.2 100 9.2 100 9.3 13 10.5 13

Spectra of 4-36: ¹H NMR (400 MHz, D₂O) δ ppm 7.33-7.40 (m, 2H), 5.65 (brd, J=5.72 Hz, 1H), 5.44-5.52 (m, 1H), 5.33 (br d, J=17.17 Hz, 2H),5.18-5.26 (m, 1H), 4.00 (br dd, J=18.06, 7.81 Hz, 1H), 3.45 (br d,J=18.84 Hz, 1H), 2.30 (s, 3H), 1.90 (q, J=7.27 Hz, 2H), 0.87 (t, J=7.27Hz, 3H). ¹⁹F NMR (376 MHz, D₂O) δ ppm −106.41. LCMS (ESI+) m/z: [MH]⁺calcd for C₂₃H₂₁FN₃O₄ ⁺: 422.1, found: 422.0. SFC (RT=1.208 min).

Spectra of 4-37: ¹H NMR (400 MHz, D₂O) δ ppm 7.43 (s, 1H), 7.24 (br d,J=11.26 Hz, 1H), 5.58 (br dd, J=8.00, 3.25 Hz, 1H), 5.54 (d, J=16.26 Hz,1H), 5.37-5.45 (m, 2H), 5.22 (br d, J=19.01 Hz, 1H), 4.05 (br dd,J=18.01, 8.13 Hz, 1H), 3.52-3.61 (m, 1H), 2.36 (s, 3H), 1.95 (q, J=7.30Hz, 2H), 0.93 (t, J=7.38 Hz, 3H). ¹⁹F NMR (376 MHz, D₂O) δ ppm −106.53.LCMS (ESI+) m/z: [MH]⁺ calcd for C₂₃H₂₁FN₃O₄ ⁺: 422.1, found: 422.0. SFC(RT=1.252 min).

Example 5 Synthesis of(1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (5-47) and(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (5-48) (FIG. 16)

3-Bromo-5-fluoro-4-methyl-aniline (1-3)

To a solution of 1-bromo-3-fluoro-2-methyl-5-nitro-benzene (1-2) (100 g,427 mmol, 1.0 equiv) in ethyl acetate (1.50 L) was added Pt/C (10 wt %,10.0 g) under argon atmosphere. After the resulting suspension wasdegassed under vacuum and purged with H₂ three times, it was stirredunder H₂ (15 psi) at 60° C. for 4 h. Then, the H₂ atmosphere wasexchanged with argon, the combined reaction mixtures were filteredthrough a pad of celite, and the filter cake washed with ethyl acetate(10.0 L). (Eighteen additional reactions were set up as described aboveand all nineteen reaction mixtures were combined). The combinedfiltrates were concentrated under reduced pressure to give3-bromo-5-fluoro-4-methyl-aniline (1-3) (1.8 kg, purity 71%, 77% yield).¹H NMR (400 MHz, DMSO-d₆) δ ppm 6.79 (s, 1H), 6.50 (dd, J=11.74, 2.08Hz, 1H), 2.10 (d, J=2.08 Hz, 3H). ¹⁹F NMR (376 MHz, CD₃OD) δ=−112.13.LCMS (ESI+) m/z: [MH]⁺ calcd for C₇H₈BrFN⁺: 205.0, found: 205.8.

N-(3-Bromo-5-fluoro-4-methyl-phenyl)acetamide (1-4)

To a solution of 3-bromo-5-fluoro-4-methyl-aniline (1-3) (300 g, 1.47mol, 1.0 equiv) in ethyl acetate (4.50 L) was added triethylamine (420mL, 3.02 mol, 2.1 equiv) and acetic anhydride (179 mL, 1.91 mol, 1.3equiv), and the mixture was stirred at 15° C. for 12 h. (Five additionalvials were set up as described above and all six reaction mixtures werecombined). The combined mixtures were quenched with saturated NH₄Clsolution (15.0 L) and extracted with ethyl acetate (3×5.0 L). Thecombined organic layers were washed with brine (8.0 L), dried overNa₂SO₄, concentrated, the residue dissolved in dichloromethane (2.00 L)and purified by column chromatography on silica gel eluting with 15% ofethyl acetate in petroleum ether to giveN-(3-bromo-5-fluoro-4-methyl-phenyl)acetamide (1-4) (1.2 kg, 55% yield).¹H NMR (400 MHz, CD₃OD) δ=7.59 (t, J=1.5 Hz, 1H), 7.41 (dd, J=2.0, 11.6Hz, 1H), 2.26 (d, J=2.2 Hz, 3H), 2.11 (s, 3H). ¹⁹F NMR (376 MHz, CD₃OD)δ=−112.95. LCMS (ESI+) m/z: [MH]⁺ calcd for C₉H₁₀BrFNO⁺: 247.0, found:247.8.

(E)-4-(5-Acetamido-3-fluoro-2-methyl-phenyl)but-3-enoic acid (5-38)

To a mixture of N-(3-bromo-5-fluoro-4-methyl-phenyl)acetamide (1-4) (200g, 813 mmol, 1.0 equiv) in tetrahydrofuran (1.00 L) and water (200 mL)were added diisopropylethyl amine (566 mL, 3.25 mol, 4.0 equiv),tris-o-tolylphosphane (49.5 g, 163 mmol, 0.20 equiv), but-3-enoic acid(168 g, 1.95 mol, 2.4 equiv) and Pd(OAc)₂ (18.2 g, 81.3 mmol, 0.10equiv) under N₂, and the reaction mixture was stirred at 75° C. for 16h. (Five additional reactions were set up as described above and all sixreaction mixtures were combined). The combined reaction mixtures werediluted with water (2.0 L), adjusted to pH=2 by addition of 3 N HCl, themixture filtered through a pad of Celite, and the filtrate cake washedwith ethyl acetate (4.0 L). The mixture was extracted with water (4.0L), the aqueous phase was extracted with ethyl acetate (3×1.80 L). Thecombined organic layers were washed with brine (4.0 L), dried overanhydrous Na₂SO₄, filtered, concentrated, and the residue purified bycolumn chromatography on silica gel eluting with 70% ethyl acetate inpetroleum ether to give(E)-4-(5-acetamido-3-fluoro-2-methyl-phenyl)but-3-enoic acid (5-38) (400g, 49% yield). ¹H NMR (400 MHz, DMSO-d₆) δ=12.32 (br s, 1H), 10.02 (s,1H), 7.48 (ddd, J=1.6, 8.3, 12.0 Hz, 1H), 7.39 (s, 1H), 7.08-6.98 (m,1H), 6.94-6.83 (m, 1H), 6.75-6.50 (m, 1H), 6.13 (td, J=7.2, 15.7 Hz,1H), 5.69 (d, J=15.5 Hz, 1H), 3.59 (ddd, J=2.5, 4.1, 6.5 Hz, 1H),3.56-3.46 (m, 1H), 3.31-3.21 (m, 1H), 2.16-2.06 (m, 3H), 2.05-1.99 (m, 3H). ¹⁹F NMR (376 MHz, DMSO-d₆) δ=−115.3. LCMS (ESI+) m/z: [MH]⁺ calcdfor C₁₃H₁₅FNO₃ ⁺: 252.1, found: 252.0.

4-(5-Acetamido-3-fluoro-2-methyl-phenyl)butanoic acid (5-39)

To a solution of (E)-4-(5-acetamido-3-fluoro-2-methyl-phenyl)but-3-enoicacid (5-38) (100 g, 398 mmol, 1.0 equiv) in methanol (1.50 L) was addedPd/C (30.0 g, 39.8 mmol, 10 wt %, 0.10 equiv) under argon atmosphere.The resulting suspension was degassed under vacuum and purged with H₂three times and stirred under H₂ (15 psi) at 35° C. for 12 h. (Threeadditional reactions were set up as described above and all fourreaction mixtures were combined). After the H₂ atmosphere was replacedwith argon, the combined mixtures filtered through a pad of celite, andthe filter cake washed with methanol (8 L). The combined filtrates wereconcentrated under reduced pressure to give4-(5-acetamido-3-fluoro-2-methyl-phenyl)butanoic acid (5-39) (330 g, 82%yield), which was used directly in next step without purification. ¹HNMR (400 MHz, DMSO-d₆) δ=12.77-11.04 (m, 1H), 9.98 (s, 1H), 7.42 (dd,J=1.7, 12.2 Hz, 1H), 7.04 (s, 1H), 2.59-2.53 (m, 2H), 2.27 (t, J=7.2 Hz,2H), 2.09 (d, J=1.8 Hz, 3H), 2.01 (s, 3H), 1.71 (quin, J=7.5 Hz, 2H).¹⁹F NMR (376 MHz, DMSO-d₆) δ=−115.64. LCMS (ESI+) m/z: [MH]⁺ calcd forC₁₃H₁₇FNO₃ ⁺: 254.1, found: 254.0.

N-(7-Fluoro-8-methyl-4-oxo-tetralin-5-yl)acetamide (5-40)

To a solution of 4-(5-acetamido-3-fluoro-2-methyl-phenyl)butanoic acid(5-39) (110 g, 434 mmol, 1.0 equiv) in trifluoroacetic acid (330 mL) wasadded trifluoroacetic anhydride (121 mL, 869 mmol, 2.0 equiv) at 0° C.under N₂, and the mixture stirred at 15° C. for 15 h. (Two additionalreactions were set up as described above and all three reaction mixtureswere combined). The combined reaction mixtures were poured into 50%acetonitrile aqueous solution (6.0 L) at 0° C. and stirred at 0° C. for0.5 h. The resulting suspension was adjusted to pH=7 with 25% NaOHaqueous solution at 0° C. The mixture was filtered, and the residewashed with water (1.00 L), methyl tert-butyl ester (2.00 L) and thendried under high vacuum. This residue was triturated with methyl methyltert-butyl ester (600 mL) and filtered to giveN-(7-fluoro-8-methyl-4-oxo-tetralin-5-yl)acetamide (5-40) (300 g, 88%yield). ¹H NMR (400 MHz, DMSO-d₆) δ=12.18 (s, 1H), 8.28 (d, J=13.2 Hz,1H), 2.89 (t, J=6.1 Hz, 2H), 2.68-2.60 (m, 2H), 2.18-2.08 (m, 6H), 1.99(quin, J=6.4 Hz, 2H). ¹⁹F NMR (376 MHz, DMSO-d₆) δ=−103.89. LCMS (ESI+)m/z: [MH]⁺ calcd for C₁₃H₁₅FNO₂ ⁺: 236.1, found: 236.0.

N-(7-Fluoro-3-hydroxy-8-methyl-4-oxo-tetralin-5-yl)acetamide (5-41)

To a solution of N-(7-fluoro-8-methyl-4-oxo-tetralin-5-yl)acetamide(5-40) (75.0 g, 319 mmol, 1.0 equiv) in methanol (1.20 L) was added asolution of KOH (53.7 g, 956 mmol, 3 equiv) in methanol (600 mL) and(diacetoxyiodo)benzene (113 g, 351 mmol, 1.1 equiv) at 0° C. underargon, and the mixture stirred at 15° C. for 3 h. (Three additionalreactions were set up as described above and all four reaction mixtureswere combined). The combined mixtures were adjusted to pH ˜4 by additionof 1 N HCl, concentrated to remove most of methanol at 35° C., and themixture extracted with dichloromethane (3×1.0 L). The combined organiclayers were washed with brine, dried over Na₂SO₄, filtered, and theresidue purified by silica gel chromatography (petroleum ether/ethylacetate=5/1 to 4/1) to giveN-(7-fluoro-3-hydroxy-8-methyl-4-oxo-tetralin-5-yl)acetamide (5-41) (210g, 66% yield). ¹H NMR (400 MHz, DMSO-d₆) δ=11.92 (s, 1H), 8.26 (d,J=13.1 Hz, 1H), 5.90-4.94 (m, 1H), 4.28 (dd, J=5.0, 12.7 Hz, 1H),3.07-2.97 (m, 1H), 2.96-2.84 (m, 1H), 2.29-2.20 (m, 1H), 2.16 (s, 3H),2.10 (d, J=1.2 Hz, 3H), 1.95-1.82 (m, 1H). ¹⁹F NMR (377 MHz, DMSO-d₆)δ=−104.3. LCMS (ESI+) m/z: [MH]⁺ calcd for C₁₃H₁₅FNO₃ ⁺: 252.1, found:252.0.

N-(3-Allyloxy-7-fluoro-8-methyl-4-oxo-tetralin-5-yl)acetamide (5-42)

To a solution ofN-(7-fluoro-3-hydroxy-8-methyl-4-oxo-tetralin-5-yl)acetamide (5-41)(70.0 g, 279 mmol, 1.0 equiv) in acetonitrile (1.40 L) was added Ag₂O(129 g, 557 mmol, 2.0 equiv) and a solution of 3-iodoprop-1-ene (140 g,836 mmol, 76.3 mL, 3.0 equiv) in acetonitrile (280 mL) at 15° C., andthe mixture stirred at 40° C. for 5 h. (Two additional reactions wereset up as described above and all three reaction mixtures werecombined). The combined mixtures were filtered through a pad of Celite,and the filter cake washed with dichloromethane (5.00 L). The combinedfiltrates were concentrated, and the residue purified by silica gelchromatography (petroleum ether/ethyl acetate=9/1-4/1) to give amaterial. This material was triturated with methyl tert-butyl ester (500mL) and filtered to affordN-(3-allyloxy-7-fluoro-8-methyl-4-oxo-tetralin-5-yl)acetamide (5-42)(198 g, 81% yield). ¹H NMR (400 MHz, DMSO-d₆) δ=11.81 (s, 1H), 8.27 (d,J=13.1 Hz, 1H), 5.93 (tdd, J=5.3, 10.5, 17.2 Hz, 1H), 5.29 (qd, J=1.8,17.2 Hz, 1H), 5.16 (dd, J=1.7, 10.5 Hz, 1H), 4.32-4.06 (m, 3H),3.12-2.83 (m, 2H), 2.32-2.23 (m, 1H), 2.17 (s, 3H), 2.11 (d, J=1.5 Hz,3H), 2.09-1.98 (m, 1H). ¹⁹F NMR (376 MHz, DMSO-d₆) δ=−104.2. LCMS (ESI+)m/z: [MH]⁺ calcd for C₁₆H₁₉FNO₃ ⁺: 292.1, found: 291.9.

N-[7-Fluoro-8-methyl-4-oxo-3-(2-oxoethoxy)tetralin-5-yl]acetamide (5-43)

A mixture ofN-(3-allyloxy-7-fluoro-8-methyl-4-oxo-tetralin-5-yl)acetamide (5-42)(42.0 g, 144 mmol, 1.0 equiv) in dichloromethane (840 mL) and methanol(420 mL) was cooled down to −70° C., ozone (6.92 g, 144 mmol, 1.0 equiv)was bubbled into the mixture for 60 min, followed by bubbled with 02 for30 min. Then, methylsulfanylmethane (26.5 mL, 360 mmol, 2.5 equiv) wasadded to the mixture at −70° C. and it was warmed up to 15° C. andstirred at 15° C. for 1 h. (Three additional reactions were set up asdescribed above and all four reaction mixtures were combined). Thecombined reaction mixtures were quenched with water (5.0 L) andextracted with dichloromethane (3×1.0 L). The combined organic layerswere washed with brine, dried over Na₂SO₄, filtered and concentrated togive N-[7-fluoro-8-methyl-4-oxo-3-(2-oxoethoxy)tetralin-5-yl]acetamide(5-43) (168 g, 59% yield), which was used directly for the next stepwithout further purification. ¹H NMR (400 MHz, CDCl₃) δ=11.94 (br s,1H), 9.80 (s, 1H), 8.44 (d, J=12.7 Hz, 1H), 4.55-4.30 (m, 1H), 3.85-3.69(m, 2H), 3.51-3.48 (m, 1H), 3.18-3.03 (m, 1H), 2.96-2.78 (m, 1H),2.52-2.34 (m, 1H), 2.26-2.21 (m, 3H), 2.14 (br dd, J=1.7, 3.9 Hz, 3H).¹⁹F NMR (376 MHz, CDCl₃) δ=−100.7. LCMS (ESI+) m/z: [MH]⁺ calcd forC₁₅H₁₇FNO₄ ⁺: 294.1, found: 293.9.

N-[7-Fluoro-3-(2-hydroxyethoxy)-8-methyl-4-oxo-tetralin-5-yl]acetamide(5-44)

To a solution ofN-[7-fluoro-8-methyl-4-oxo-3-(2-oxoethoxy)tetralin-5-yl]acetamide (5-43)(42 g, 85.9 mmol, 60% purity, 1.0 equiv) in THF (850 mL) and H₂O (425mL) was added NaBH₄ (975 mg, 25.8 mmol, 0.30 equiv) portion wise at 0°C., the mixture was stirred at 0° C. for 10 min, and quenched with coldwater (2.0 L). (Three additional reactions were set up as describedabove and all four reaction mixtures were combined). The combinedreaction mixtures were extracted with dichloromethane (3×1.0 L), organiclayers washed with brine, dried over Na₂SO₄, filtered, concentrated andthe residue purified by silica gel chromatography (petroleum ether/ethylacetate=1/1-1/3) to giveN-[7-fluoro-3-(2-hydroxyethoxy)-8-methyl-4-oxo-tetralin-5-yl]acetamide(5-44) (70 g, 61% yield). ¹H NMR (400 MHz, CDCl₃) δ=11.96 (br s, 1H),8.45 (d, J=12.7 Hz, 1H), 4.06 (dd, J=4.6, 12.0 Hz, 1H), 4.01-3.62 (m,4H), 3.11 (td, J=4.6, 17.6 Hz, 1H), 3.03-2.79 (m, 2H), 2.45-2.36 (m,1H), 2.24 (s, 3H), 2.15 (d, J=1.5 Hz, 4H). ¹⁹F NMR (376 MHz, CDCl₃)δ=−100.9. LCMS (ESI+) m/z: [MH]⁺ calcd for C₁₅H₁₉FNO₄ ⁺: 296.1, found:296.1.

8-Amino-6-fluoro-2-(2-hydroxyethoxy)-5-methyl-tetralin-1-one (5-45)

To a solution ofN-[7-fluoro-3-(2-hydroxyethoxy)-8-methyl-4-oxo-tetralin-5-yl]acetamide(5-44) (21.0 g, 71.0 mmol, 1.0 equiv) in methanol (400 mL) was added HCl(2 N, 630 mL, 18 equiv) under argon, the mixture was stirred at 15° C.for 18 h and adjusted to pH=7˜8 by addition of saturated NaHCO₃. (Threeadditional reactions were set up as described above and all fourreaction mixtures were combined). The combined reaction mixtures wereextracted with ethyl acetate (3×2.0 L), organic layers washed withbrine, dried over Na₂SO₄, filtered and concentrated. The residue wastriturated with methyl tert-butyl ester/dichloromethane (1:2, 300 mL)and filtered to give8-amino-6-fluoro-2-(2-hydroxyethoxy)-5-methyl-tetralin-1-one (5-45) (42g, 81% yield). ¹H NMR (400 MHz, CD₃OD) δ=6.30 (d, J=12.3 Hz, 1H), 4.06(dd, J=4.4, 11.2 Hz, 1H), 3.83-3.67 (m, 4H), 3.04 (td, J=4.9, 17.5 Hz,1H), 2.85-2.73 (m, 1H), 2.40-2.30 (m, 1H), 2.09-1.93 (m, 4H). ¹⁹F NMR(376 MHz, CD₃OD) δ=−108.7. LCMS (ESI+) m/z: [MH]⁺ calcd for C₁₃H₁₇FNO₃⁺: 254.1, found: 254.1.

(9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (5-46)

To a mixture of8-amino-6-fluoro-2-(2-hydroxyethoxy)-5-methyl-3,4-dihydronaphthalen-1(2H)-one (5-45) (10.5 g, 41.4 mmol, 1.0 equiv) and(S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-A]indolizine-3,6,10(4H)-trione (1-13) (12.0 g, 45.6 mmol, 1.1 equiv) in toluene (525 mL)was added o-cresol (31.5 mL, 303 mmol, 7.3 equiv) and pyridin-1-ium4-methylbenzenesulfonate (1.56 g, 6.23 mmol, 0.15 equiv) at 120° C.under argon, and the mixture was stirred at 120° C. for 13 h. (Threeadditional reactions were set up as described above and four reactionmixtures were combined). The combined reaction mixtures wereconcentrated under reduced pressure, and the residue purified by columnchromatography on silica gel, eluting with 13% of methanol in ethylacetate to give(9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (5-46) (34.2 g, 43% yield). ¹H NMR (400 MHz, DMSO-D₆) δppm 7.58 (dd, J=10.8, 1.91 Hz, 1H), 7.23 (d, J=6.8 Hz, 1H), 6.49 (s,1H), 5.40 (s, 2H), 4.95-5.24 (m, 2H), 4.88 (dt, J=8.4, 4.4 Hz, 1H),4.69-4.81 (m, 1H), 3.78-3.91 (m, 1H), 3.56-3.76 (m, 3H), 3.10 (d, J=16.4Hz, 1H), 2.77-2.92 (m, 1H), 2.33-2.45 (m, 1H), 2.21 (s, 3H), 1.83-2.04(m, 3H), 0.77-1.00 (m, 3H). ¹⁹F NMR (376 MHz, DMSO-D₆) δ ppm −111.57.LCMS (ESI+) m/z: [M+H]⁺ calcd for C₂₆H₂₆FN₂O₆ ⁺: 481.2, found: 481.0.

(1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (5-47) and(1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (5-48)

(9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (5-46) (31.0 g, 64.3 mmol, 1.0 equiv) was separated bychiral SFC (Instrument: Waters SFC350 preparative SFC; Column: DAICELCHIRALCEL OD (250 mm*50 mm, 10 um); Mobile phase: A for CO₂ and B forMeOH; Gradient: B %=60% isocratic elution mode; Flow rate: 200 g/min;Wavelength: 220 nm; Column temperature: 40 degrees centigrade; Systemback pressure: 100 bar) to afford(1R,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (5-47) (13.5 g, 43% yield) (compound 5-47 may be theopposite enantiomer of that depicted) and(1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (5-48) (10.0 g, 32% yield) (compound 5-48 may be theopposite enantiomer of that depicted). Note: the stereochemistry isarbitrarily assigned.

Spectra of 5-47: ¹H NMR (400 MHz, DMSO-D₆) δ ppm 7.64 (d, J=10.88 Hz,1H), 7.25 (s, 1H), 6.50 (s, 1H), 5.13-5.52 (m, 4H), 4.95 (br dd, J=8.25,3.75 Hz, 1H), 4.74 (br t, J=5.00 Hz, 1H), 3.84 (dt, J=9.72, 4.96 Hz,1H), 3.59-3.76 (m, 3H), 3.10-3.21 (m, 1H), 2.84-2.99 (m, 1H), 2.23-2.46(m, 4H), 2.04 (m, 1H), 1.87 (m, 2H), 0.88 (t, J=7.32 Hz, 3H). ¹⁹F NMR(376 MHz, DMSO-D₆) δ ppm −111.59. LCMS (ESI+) m/z: [M+H]⁺ calcd forC₂₆H₂₆FN₂O₆ ⁺: 481.2, found: 481.0. Chiral SFC: RT=1.48 min.

Spectra of 5-48: ¹H NMR (400 MHz, DMSO-D₆) δ ppm 7.74 (d, J=10.97 Hz,1H), 7.30 (s, 1H), 6.51 (s, 1H), 5.31-5.46 (m, 4H), 5.03 (dd, J=8.11,3.70 Hz, 1H), 4.74 (t, J=5.36 Hz, 1H), 3.81-3.89 (m, 1H), 3.59-3.75 (m,3H), 3.21 (dt, J=17.02, 5.38 Hz, 1H), 2.94-3.07 (m, 1H), 2.31-2.45 (m,4H), 2.08 (m, 1H), 1.88 (dt, J=13.23, 6.62 Hz, 2H), 0.89 (t, J=7.33 Hz,3H). ¹⁹F NMR (376 MHz, DMSO-D₆) δ ppm −111.53. LCMS (ESI+) m/z: [M+H]⁺calcd for C₂₆H₂₆FN₂O₆ ⁺: 481.2, found: 481.0. Chiral SFC: RT=1.61 min.

Example 6 Synthesis of(1R,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-34) and(1S,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-35) (FIG. 17)

2-(Allyloxy)-8-amino-6-fluoro-5-methyl-3,4-dihydronaphthalen-1 (2H)-one(6-49)

To a mixture ofN-(7-(allyloxy)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide(5-42) (600 mg, 2.06 mmol, 1.0 equiv) in methanol (6.0 mL) was addedHCl/Methanol (6.0 mL, 4 M, 11.65 equiv) at 0° C. under argon, themixture was stirred at 25° C. for 2 h, cooled to 0° C. and the pHadjusted to 8 by addition of saturated NaHCO₃. It was extracted withdichloromethane (3×40 mL), combined organic layers washed with brine,dried over Na₂SO₄, filtered, concentrated under reduced pressure, andthe residue purified by column chromatography on silica gel eluting with10% of ethyl acetate in petroleum ether to give2-(allyloxy)-8-amino-6-fluoro-5-methyl-3,4-dihydronaphthalen-1 (2H)-one(6-49) (415 mg, 81% yield). ¹H NMR (400 MHz, CDCl₃) δ ppm 6.24-6.68 (m,2H), 6.20 (d, J=11.62 Hz, 1H), 5.91-6.03 (m, 1H), 5.33 (dq, J=17.24,1.63 Hz, 1H), 5.20 (dq, J=10.38, 1.35 Hz, 1H), 4.36 (m, 1H), 4.17 (m,1H), 3.97 (dd, J=10.15, 4.16 Hz, 1H), 3.01 (dt, J=17.45, 5.33 Hz, 1H),2.75 (m, 1H), 2.27 (dq, J=13.17, 5.02 Hz, 1H), 2.09-2.19 (m, 1H), 2.04(d, J=1.71 Hz, 3H). ¹⁹F NMR (376 MHz, CDCl₃) δ ppm −106.28. LCMS (ESI+)m/z: [MH]⁺ calcd for C₁₄H₁₇FNO₂ ⁺: 250.1, found: 250.1.

(9S)-1-(Allyloxy)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (6-50)

To a mixture of2-(allyloxy)-8-amino-6-fluoro-5-methyl-3,4-dihydronaphthalen-1 (2H)-one(6-49) (200 mg, 0.802 mmol, 1.0 equiv), and(S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (232 mg, 0.883 mmol, 1.1 equiv) in toluene (10 mL) at120° C. were added o-cresol (0.609 mL, 5.86 mmol, 7.3 equiv) andpyridin-1-ium 4-methylbenzenesulfonate (30.2 mg, 0.120 mmol, 0.15 equiv)under argon, the mixture was stirred at 120° C. for 32 h, concentratedunder reduced pressure, and the residue purified by columnchromatography on silica gel eluting with 55% of ethyl acetate inpetroleum ether to give(9S)-1-(allyloxy)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (6-50) (100 mg, 26% yield). ¹H NMR (400 MHz, CDCl₃) δ ppm7.58-7.67 (m, 2H), 6.03-6.16 (m, 1H), 5.74 (dd, J=16.26, 1.83 Hz, 1H),5.39-5.52 (m, 2H), 5.21-5.36 (m, 3H), 4.94 (m, 1H), 4.38-4.47 (m, 1H),4.26 (m, 1H), 3.87 (d, J=19.32 Hz, 1H), 3.23-3.36 (m, 1H), 2.99 (br t,J=13.02 Hz, 1H), 2.45-2.57 (m, 1H), 2.40 (br s, 3H) 2.11-2.25 (m, 1H),1.82-1.98 (m, 2H), 1.04 (td, J=7.37, 2.87 Hz, 3H). ¹⁹F NMR (376 MHz,CDCl₃) δ ppm −110.35. LCMS (ESI+) m/z: [MH]⁺ calcd for C₂₇H₂₆FN₂O₅ ⁺:477.2, found: 477.2.

(9S)-9-Ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-33)

To a mixture of(9S)-1-(allyloxy)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydro-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (6-50) (300 mg, 0.63 mmol, 1.0 equiv) in tetrahydrofuran(15 mL) was added ZnCl₂ (110 mg, 0.82 mmol, 1.3 equiv) at 25° C. underargon, and 0.25 h later Pd(PPh₃)₄ (58.0 mg, 0.157 mmol, 0.25 equiv), andanother 0.25 h later Bu₃SnH (3.33 mL, 12.6 mmol, 20 equiv), and themixture was stirred at 50° C. for 1.5 h. It was concentrated underreduced pressure, and the residue purified by column chromatography onsilica gel eluting with 5% of methanol in ethyl acetate to give aresidue, which was further purified by prep-HPLC to afford(9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-33) (90.0 mg, 32% yield). ¹H NMR (400 MHz, DMSO-D₆) δppm 7.72 (d, J=10.97 Hz, 1H), 7.30 (d, J=2.27 Hz, 1H), 6.50 (d, J=0.72Hz, 1H), 5.98 (dd, J=5.90, 2.92 Hz, 1H), 5.36-5.46 (m, 3H), 5.23-5.32(m, 1H), 5.14 (dt, J=9.60, 4.86 Hz, 1H), 3.23 (dt, J=16.90, 4.60 Hz,2H), 2.96-3.06 (m, 1H), 2.35 (s, 3H), 1.94-2.06 (m, 1H), 1.80-1.93 (m,2H), 0.88 (td, J=7.30, 1.49 Hz, 3H). ¹⁹F NMR (376 MHz, DMSO-D₆) δ ppm−111.78. LCMS (ESI+) m/z: [MH]⁺ calcd for C₂₄H₂₂FN₂O₅ ⁺: 437.2, found:437.0.

Prep-HPLC Method:

-   -   Instrument: Gilson 281 semi-preparative HPLC system    -   Mobile phase: A: H₂O; B: MeOH    -   Column: Phenomenex Luna 80*30 mm*3 um    -   Flow rate: 25 mL/min    -   Monitor wavelength: 220&254 nm

Time B % 0.0 45 8.0 70 8.1 70 8.2 100 10.2 100 10.3 45 11.5 45

(1R,9S)-9-Ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-34) and(1S,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-35)

(9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-33) (90.0 mg, 0.206 mmol, 1.0 equiv) was separated bychiral SFC (Instrument: Waters SFC80 preparative SFC; Column: DAICELCHIRALCEL OD(250 mm*30 mm, 10 um); Mobile phase: A for CO₂ and B forEtOH; Gradient: B %=55% isocratic elution mode; Flow rate: 60 g/min;Wavelength: 220 nm; Column temperature: 40° C.; System back pressure:100 bar.) to afford(1R,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-34) (26.1 mg, 29% yield) (compound 3-34 may be theopposite enantiomer of that depicted) and(1S,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (3-35) (25.1 mg, 28% yield) (compound 3-35 may be theopposite enantiomer of that depicted). Note: The stereochemistry for twoproducts are arbitrarily assigned.

Spectra of 3-34: ¹H NMR (400 MHz, DMSO-D₆) δ ppm 7.73 (d, J=10.97 Hz,1H), 7.30 (s, 1H), 6.50 (s, 1H), 5.98 (d, J=5.84 Hz, 1H), 5.37-5.47 (m,3H), 5.26-5.35 (m, 1H), 5.10-5.20 (m, 1H), 3.20-3.30 (m, 2H), 2.97-3.07(m, 1H), 2.35 (s, 3H), 1.95-2.05 (m, 1H), 1.78-1.94 (m, 2H), 0.88 (t,J=7.33 Hz, 3H). ¹⁹F NMR (376 MHz, DMSO-D₆) δ ppm −111.78. LCMS (ESI+)m/z: [MH]⁺ calcd for C₂₄H₂₂FN₂O₅: 437.2, found: 437.0. Chiral SFC:RT=1.43 min.

Spectra of 3-35: ¹H NMR (400 MHz, DMSO-D₆) δ ppm 7.73 (d, J=11.09 Hz,1H), 7.30 (s, 1H), 6.50 (s, 1H), 5.97 (d, J=5.84 Hz, 1H), 5.34-5.48 (m,3H), 5.21-5.32 (m, 1H), 5.09-5.20 (m, 1H), 3.20-3.24 (m, 2H), 2.96-3.08(m, 1H), 2.35 (s, 3H), 1.99 (d, J=9.54 Hz, 1H), 1.80-1.93 (m, 2H), 0.89(t, J=7.09 Hz, 3H). ¹⁹F NMR (376 MHz, DMSO-D₆) δ ppm −111.80. LCMS(ESI+) m/z: [MH]⁺ calcd for C₂₄H₂₂FN₂O₅: 437.2, found: 437.0. ChiralSFC: RT=1.54 min.

Example 7(1S,10S)-1-amino-10-ethyl-6-fluoro-10-hydroxy-5-methyl-3,4,13,16-tetrahydro-1H-cyclohepta[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-11,14(2H,10H)-dione methanesulfonate (7-59) and(1R,10S)-1-amino-10-ethyl-6-fluoro-10-hydroxy-5-methyl-3,4,13,16-tetrahydro-1H-cyclohepta[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-11,14(2H,10H)-dione methanesulfonate (7-60) (FIG. 18)

5-(5-Acetamido-3-fluoro-2-methylphenyl)pent-4-enoic acid (7-51)

To a solution of N-(3-bromo-5-fluoro-4-methyl-phenyl)acetamide (1-4)(50.0 g, 203 mmol, 1.0 equiv) in THF (250 mL) and H₂O (50.0 mL) wasadded pent-4-enoic acid (49.9 mL, 487 mmol, 2.4 equiv), diisopropylethylamine (155 mL, 894 mmol, 4.4 equiv), tris-o-tolylphosphane (12.4 g, 40.6mmol, 0.20 equiv) and palladium (II) diacetae (4.56 g, 20.3 mmol, 0.10equiv) under N₂ atmosphere, the mixture was stirred at 75° C. for 24 hand cooled down to 30° C. (Three additional reactions were set up asdescribed above and all four reaction mixtures were combined). Thecombined mixtures were filtered through a pad of celite, and the filtercake washed with water (2.0 L) and ethyl acetate (3.0 L). The mixturewas adjusted to pH 4-5 with HCl solution (5 N), extracted with ethylacetate (3×800 mL), organic layers dried over Na₂SO₄, concentrated, andthe residue purified by silica gel flash column chromatography elutingwith 30% of ethyl acetate in petroleum ether to give5-(5-acetamido-3-fluoro-2-methylphenyl) pent-4-enoic acid (7-51) (0.20kg, 83% yield). ¹H NMR (400 MHz, DMSO-d₆) δ=12.19 (br s, 1H), 9.99 (s,1H), 7.51-7.28 (m, 1H), 7.00 (s, 1H), 6.17-5.36 (m, 2H), 3.35-3.24 (m,2H), 3.19-2.93 (m, 2H), 2.07 (s, 3H), 2.01 (s, 3H)¹⁹F NMR (376 MHz,DMSO-d₆) δ ppm −115.96.

5-(5-Acetamido-3-fluoro-2-methylphenyl) pentanoic acid (7-52)

To a mixture of 5-(5-acetamido-3-fluoro-2-methylphenyl)pent-4-enoic acid(7-51) (50.0 g, 170 mmol, 90% purity, 1.0 equiv) in methanol (500 mL)was added Pd/C (25.0 g, 170 mmol, 10 wt %, 1.0 equiv) under argonatmosphere. After the mixture was vacuumed and filled with H₂ threetimes, it stirred at 25° C. for 12 h under H₂ (15 Psi). (Threeadditional reactions were set up as described above and all fourreaction mixtures were combined). After the H₂ atmosphere was replacedwith argon, it was filtered through a pad of celite, filter cake washedwith methanol (6.00 L), filtrates concentrated to give5-(5-acetamido-3-fluoro-2-methylphenyl) pentanoic acid (7-52) (190 g,94% yield), which was used directly in the next step directly withoutfurther purification. ¹H NMR (400 MHz, DMSO-d₆) δ=9.97 (s, 1H), 7.41(dd, J=1.7, 12.2 Hz, 1H), 7.03 (s, 1H), 2.54 (br t, J=7.4 Hz, 2H), 2.23(t, J=7.0 Hz, 2H), 2.08 (d, J=1.7 Hz, 3H), 2.01 (s, 3H), 1.59-1.43 (m,4H). ¹⁹F NMR (376 MHz, CDCl₃) δ ppm −115.806. LCMS (ESI+) m/z: [MH]⁺calcd for C₁₄H₁₉FNO₃ ⁺: 268.1, found: 268.0.

N-(3-fluoro-4-methyl-9-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulen-1-yl)acetamide(7-53)

A mixture of 5-(5-acetamido-3-fluoro-2-methylphenyl)pentanoic acid(7-52) (38.0 g, 142 mmol, 1.0 equiv) in polyphosphoric acid (500 mL) wasstirred at 110° C. for 3 h. (Four additional reactions were set up asdescribed above and all five reaction mixtures were combined). Thecombined reaction mixtures were slowly poured into stirred ice water(10.0 L), the pH adjusted to 7 with saturated NaHCO₃ solution, themixture extracted with ethyl acetate (3×1.00 L). The combined organiclayers were washed with brine, dried over anhydrous Na₂SO₄, filtered,concentrated under reduced pressure, and the residue purified by silicagel flash column chromatography eluting with 15% of ethyl acetate inpetroleum ether to giveN-(3-fluoro-4-methyl-9-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulen-1-yl)acetamide(7-53) (91 g, 64% yield). ¹H NMR (400 MHz, DMSO-d₆) δ=9.83 (s, 1H), 7.19(d, J=11.6 Hz, 1H), 2.69 (t, J=6.3 Hz, 2H), 2.58-2.52 (m, 2H), 2.16 (d,J=2.0 Hz, 3H), 1.95 (s, 3H), 1.74-1.58 (m, 4H). ¹⁹F NMR (376 MHz,DMSO-d₆) δ ppm −111.49. LCMS (ESI+) m/z: [MH]⁺ calcd for C₁₄H₁₇FNO₂ ⁺:250.12, found: 250.0.

(Z)—N-(3-fluoro-8-(hydroxyimino)-4-methyl-9-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulen-1-yl)acetamide(7-54)

To a mixture of potassium tert-butoxide (1 M in tetrahydrofuran, 191 mL,2.1 equiv) in tetrahydrofuran (194 mL), ethanol (31.4 mL), and n-butanol(31.4 mL), were addedN-(2-fluoro-1-methyl-5-oxo-6,7,8,9-tetrahydrobenzo[7]annulen-4-yl)acetamide(7-53) (22.7 g, 90.9 mmol, 1.0 equiv) and isopentyl nitrite (14.8 mL,109 mmol, 1.2 equiv) at 0° C., and the mixture was stirred at 20° C. for3 h. (Three additional reactions were set up as described above and allfour reaction mixtures were combined). The combined reaction mixtureswere cooled to 0° C., quenched with 0.5 N hydrochloric acid (1.00 L) andextracted with ethyl acetate (3×300 mL), organic layers washed withbrine, dried over sodium sulfate, filtered, concentrated under reducedpressure. The residue was triturated with methyl tert-butyl ester (500mL), and filtered to give(Z)—N-(3-fluoro-8-(hydroxyimino)-4-methyl-9-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulen-1-yl)acetamide(7-54) (80.0 g, 88% yield). ¹H NMR (400 MHz, DMSO-d₆) δ=12.69-11.95 (m,1H), 10.10 (s, 1H), 7.28 (d, J=11.6 Hz, 1H), 2.68 (br t, J=6.6 Hz, 2H),2.57 (t, J=6.8 Hz, 2H), 2.17 (d, J=1.8 Hz, 3H), 1.96 (s, 3H), 1.78-1.75(m, 2H)¹⁹F NMR (376 MHz, DMSO-d₆) δ ppm −107.85 , −109.67 , −110.39.LCMS (ESI+) m/z: [MH]⁺ calcd for C₁₄H₁₆FN₂O₃ ⁺: 279.1, found: 279.1.

N-(8-amino-3-fluoro-4-methyl-9-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annule-1-yl)acetamidehydrochloric acid salt (7-55)

To a mixture ofN-[(5E)-2-fluoro-5-hydroxyimino-1-methyl-6,7,8,9-tetrahydrobenzo[7]annulen-4-yl]acetamide(7-54) (20.0 g, 75.7 mmol, 1.0 equiv) in methanol (240 mL) were addedhydrochloric acid (12 M, 18.9 mL, 3.0 equiv) and Pd/C (4.00 g, 75.7mmol, 10 wt %, 1.0 equiv) under N₂ atmosphere, the mixture was vacuumedand filled with H₂ three times, and stirred at 20° C. for 3 h under H₂(15 Psi). (Three additional reactions were set up as described above andall four reaction mixtures were combined). After the H₂ atmosphere wasreplaced with argon, the reaction mixtures were combined, diluted withmethanol (800 mL), filtered through a pad of celite, filter cake washedwith methanol (10.0 L), combined filtrates concentrated under reducedpressure to dryness. The residue was triturated with methyl tert-butyether (800 mL) and filtered to giveN-(8-amino-3-fluoro-4-methyl-9-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulen-1-yl)acetamidehydrochloric acid salt (7-55) (75 g, 98% yield). ¹H NMR showed thismaterial contained about 25% de-Ac byproduct. ¹H NMR (400 MHz, DMSO-d₆)δ=10.34 (s, 1H), 8.75-8.40 (m, 3H), 7.31-7.20 (m, 1H), 4.44-4.21 (m,1H), 2.98 (dt, J=6.1, 7.9 Hz, 1H), 2.85-2.75 (m, 1H), 2.47 (br s, 1H),2.27-2.11 (m, 3H), 2.10-1.94 (m, 3H), 1.94-1.50 (m, 3H). ¹⁹F NMR (376MHz, DMSO-d₆) δ ppm −107.88, −110.63. LCMS (ESI+) m/z: [MH]⁺ calcd forC₁₄H₁₆FN₂O₃ ⁺: 265.1, found: 265.0.

N,N′-(3-fluoro-4-methyl-9-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulene-1,8-iyl)diacetamide(7-56)

To a suspension ofN-(5-amino-2-fluoro-1-methyl-6,7,8,9-tetrahydro-5H-benzo[7]annulen-4-yl)acetamidehydrochloric acid salt (7-55) (25.0 g, 87.2 mmol, 1.0 equiv) indichloromethane (225 mL) were added triethylamine (36.4 mL, 262 mmol,3.0 equiv) and acetic anhydride (9.80 mL, 105 mmol, 1.2 equiv), and themixture was stirred at 20° C. for 3 h. (Three additional reactions wereset up as described above and all four reaction mixtures were combined).The combined reaction mixtures were extracted with saturated NH₄Clsolution (2.00 L), the aqueous phase extracted with ethyl acetate (3×500mL), combined organic layers dried over Na₂SO₄, filtered, concentratedunder reduced pressure, and the residue purified by silica gel flashcolumn chromatography eluting with 30% of ethyl acetate in petroleumether to giveN,N′-(3-fluoro-4-methyl-9-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulene-1,8-diyl)diacetamide(7-56) (54.0 g, 53% yield) andN-(4-amino-2-fluoro-1-methyl-6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-yl)acetamide (7-57) (9.00 g, 10% yield).

Spectra of 7-56: ¹H NMR (400 MHz, DMSO-d₆) δ=9.61 (br s, 1H), 8.69 (brd, J=4.5 Hz, 1H), 7.69 (d, J=12.2 Hz, 1H), 4.56-4.39 (m, 1H), 2.93 (brdd, J=7.0, 12.3 Hz, 1H), 2.43 (br dd, J=10.3, 14.7 Hz, 1H), 2.14 (d,J=1.2 Hz, 3H), 2.01 (s, 4H), 1.93 (s, 4H), 1.81-1.61 (m, 2H). ¹⁹F NMR(376 MHz, DMSO-d₆) δ ppm −111.86. LCMS (ESI+) m/z: [M+H]⁺ calcd forC₁₆H₂₀FN₂O₃ ⁺: 307.1, found: 307.1.

Spectra of 7-57: ¹H NMR (400 MHz, DMSO-d₆) δ=8.25 (br d, J=7.1 Hz, 1H),6.45-6.33 (m, 3H), 4.57 (td, J=6.7, 11.5 Hz, 1H), 2.97-2.86 (m, 1H),2.81-2.70 (m, 1H), 2.07-1.97 (m, 4H), 1.95-1.88 (m, 1H), 1.84 (s, 3H),1.70-1.59 (m, 1H), 1.57-1.42 (m, 1H). ¹⁹F NMR (376 MHz, DMSO) δ ppm−110.23. LCMS (ESI+) m/z: [M+H]⁺ calcd for C₁₄H₁₈FN₂O₃ ⁺: 265.1, found:265.1.

N-(4-amino-2-fluoro-1-methyl-5-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulen-6-yl)acetamide (7-57)

To a mixture ofN,N′-(3-fluoro-4-methyl-9-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulene-1,8-diyl)diacetamide(7-56) (18.0 g, 61.6 mmol, 1.0 equiv) in methanol (180 mL) was addedhydrochloric acid/methanol (4 M, 180 mL, 11.7 equiv), and the mixturewas stirred at 25° C. for 2 h. (Three additional reactions were set upas described above and all four reaction mixtures were combined). Thecombined reaction mixtures were concentrated under reduced pressure, andthe residue extracted with saturated NaHCO₃ solution (2.00 L) and ethylacetate/methanol (10/1, 3×800 mL). The combined organic phases werewashed with brine, dried over anhydrous Na₂SO₄, filtered, concentratedunder reduced pressure, and the residue purified by silica gel flashcolumn chromatography eluting with 30% of ethyl acetate in petroleumether to giveN-(4-amino-2-fluoro-1-methyl-6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-yl)acetamide (7-57) (25 g, 54% yield). ¹H NMR (400 MHz, DMSO-d₆) δ=8.25 (brd, J=7.1 Hz, 1H), 6.45-6.33 (m, 3H), 4.57 (td, J=6.7, 11.5 Hz, 1H),2.97-2.86 (m, 1H), 2.81-2.70 (m, 1H), 2.07-1.97 (m, 4H), 1.95-1.88 (m,1H), 1.84 (s, 3H), 1.70-1.59 (m, 1H), 1.57-1.42 (m, 1H). ¹⁹F NMR (376MHz, CDCl₃) δ ppm −111.22. LCMS (ESI+) m/z: [M+H]⁺ calcd for C₁₄H₁₈FN₂O₃⁺: 265.1, found: 265.1.

N-((10S)-10-Ethyl-6-fluoro-10-hydroxy-5-methyl-11,14-dioxo-2,3,4,10,11,13,14,16-octa-hydro-1H-cyclohepta[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide(7-58)

To a mixture ofN-(4-amino-2-fluoro-1-methyl-5-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulen-6-yl)acetamide(7-57) (230 mg, 0.870 mmol, 1.0 equiv) and(S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (458 mg, 1.74 mmol, 2.0 equiv) in toluene (23 mL) wasadded pyridin-1-ium 4-methylbenzenesulfonate (87.4 mg, 0.348 mmol, 0.4equiv), and the mixture was stirred at 120° C. for 16 h in a sealedtube. The reaction mixture was concentrated under reduced pressure, andthe residue purified by silica gel flash column chromatography elutingwith 50% ethyl acetate in petroleum ether to affordN-((10S)-10-ethyl-6-fluoro-10-hydroxy-5-methyl-11,14-dioxo-2,3,4,10,11,13,14,16-octahydro-1H-cyclohepta[de]pyrano[3′,4′:6,7]indole-zino[1,2-b]quinolin-1-yl)acetamide(7-58) (240 mg, 54% yield). ¹H NMR (400 MHz, DMSO-D₆) δ=8.71 (br d,J=6.2 Hz, 1H), 7.73 (d, J=10.5 Hz, 1H), 7.29 (s, 1H), 6.52 (d, J=7.4 Hz,1H), 5.62-5.50 (m, 1H), 5.46-5.31 (m, 3H), 5.22-5.11 (m, 1H), 3.27-3.20(m, 2H), 3.17 (d, J=4.8 Hz, 1H), 2.42 (s, 3H), 2.31-2.20 (m, 1H),2.14-2.02 (m, 2H), 1.97 (s, 3H), 1.84-1.60 (m, 2H), 0.87 (q, J=7.4 Hz,3H). LCMS (ESI+) m/z: [MH]⁺ calcd for C₂₇H₂₇FN₃O₅ ⁺: 492.2, found:492.1.

(1S,10S)-1-Amino-10-ethyl-6-fluoro-10-hydroxy-5-methyl-3,4,13,16-tetrahydro-1H-cyclo-hepta[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-11,14(2H,10H)-dione methanesulfonate (7-59) and(1R,10S)-1-amino-10-ethyl-6-fluoro-10-hydroxy-5-methyl-3,4,13,16-tetrahydro-1H-cyclohepta[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-11,14(2H,10H)-dione methanesulfonate (7-60)

A mixture ofN-((10S)-10-ethyl-6-fluoro-10-hydroxy-5-methyl-11,14-dioxo-2,3,4,10,11,13,14,16-octahydro-1H-cyclohepta[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide(7-58) (160 mg, 0.101 mmol, 1.0 equiv) in 2-methoxyethanol (0.8 mL) wereadded methylcyclohexane (0.8 mL), H₂O (0.75 mL) and methanesulfonic acid(0.25 mL) under argon, the mixture was stirred at 100° C. for 8 h,concentrated, and the residue purified by prep-HPLC to afford(1S,10S)-1-amino-10-ethyl-6-fluoro-10-hydroxy-5-methyl-3,4,13,16-tetrahydro-1H-cyclohepta[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-11,14(2H,10H)-dione methanesulfonate (7-59) (5.5 mg, 2.4% yield) (compound7-59 may be the opposite enantiomer of that depicted) and(1R,10S)-1-amino-10-ethyl-6-fluoro-10-hydroxy-5-methyl-3,4,13,16-tetrahydro-1H-cyclohepta[de]pyrano[3′,4′:6,7]indolezino[1,2-b]quinoline-11,14(2H,10H)-dione methanesulfonate (7-60) (6.4 mg, 3.6% yield) (compound7-60 may be the opposite enantiomer of that depicted). Note: thestereochemistry is arbitrarily assigned.

Prep-HPLC Method:

-   -   Instrument: Gilson 281 semi-preparative HPLC system    -   Mobile phase: A: H₂O; B: MeOH    -   Column: Phenomenex Luna 80*30 mm*3 um    -   Flow rate: 25 mL/min    -   Monitor wavelength: 220&254 nm

Time B % 0.0 40 6.0 75 6.1 75 8.2 100 10.2 100 10.3 40 11.5 40

Spectra of 7-59: ¹H NMR (400 MHz, D₂O) δ ppm 7.38-7.49 (m, 2H),5.52-5.62 (m, 1H), 5.35-5.46 (m, 3H), 5.16 (t, J=4.4 Hz, 1H), 3.40-3.51(m, 1H), 2.99-3.13 (m, 1H), 2.77 (s, 6H), 2.48-2.57 (m, 2H), 2.38 (d,J=1.6 Hz, 3H), 2.22-2.33 (m, 1H), 2.06-2.20 (m, 1H), 1.94 (q, J=7.5 Hz,2H), 0.90 (t, J=7.2 Hz, 3H). ¹⁹F NMR (376 MHz, D₂O) δ ppm −111.53. LCMS(ESI+) m/z: [MH]⁺ calcd for C₂₅H₂₅FN₃O₄ ⁺: 450.2, found: 450.0.

Spectra of 7-60: ¹H NMR (400 MHz, D₂O) δ ppm 7.40-7.51 (m, 2H),5.44-5.60 (m, 2H), 5.33-5.43 (m, 2H), 5.09-5.14 (m, 1H), 3.37-3.48 (m,1H), 3.01-3.14 (m, 1H), 2.77 (s, 3H), 2.43-2.54 (m, 1H), 2.39 (s, 3H),2.27-2.35 (m, 1H), 2.06-2.23 (m, 2H), 1.95 (q, J=7.6 Hz, 2H), 0.92 (t,J=7.2 Hz, 3H). ¹⁹F NMR (376 MHz, D₂O) δ ppm −109.21. LCMS (ESI+) m/z:[MH]⁺ calcd for C₂₅H₂₅FN₃O₄ ⁺: 450.2, found: 450.0.

Example 8(1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (8-71) and(1R,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (8-72) (FIG. 19)

4-(5-Acetamido-3-fluoro-2-methylphenyl)-2-methylbut-3-enoic acid (8-61)

To a mixture of N-(3-bromo-5-fluoro-4-methyl-phenyl)acetamide (1-4)(10.0 g, 40.6 mmol, 1.0 equiv) and 2-methylbut-3-enoic acid (13.0 g, 130mmol, 3.2 equiv) in tetrahydrofuran (40 mL) and water (10 mL) were addedN-ethyl-N,N-diisopropylamine (38.2 mL, 219 mmol, 5.4 equiv),tris-o-tolylphosphane (2.47 g, 8.13 mmol, 0.2 equiv) anddiacetoxypalladium (912 mg, 4.06 mmol, 0.1 equiv) under nitrogen, andthe mixture was stirred at 75° C. for 5 h., quenched with water (50 mL)and the pH adjusted to around 3 by addition of 3 N hydrochloric acid at0° C. It was filtered through a pad of Celite, and the filtrateextracted with ethyl acetate (3×150 mL), organic layers washed withbrine, dried over Na₂SO₄, filtered, concentrated, and the residuepurified by silica gel flash column chromatography eluting with 50%ethyl acetate in petroleum ether to give4-(5-acetamido-3-fluoro-2-methylphenyl)-2-methylbut-3-enoic acid (8-61)(6.00 g, 56% yield). ¹H NMR (400 MHz, DMSO-D₆) δ ppm 12.38 (s, 1H),10.02 (d, J=4.0 Hz, 1H), 7.46-7.52 (m, 1H), 6.99-7.38 (m, 1H), 6.65-6.69(m, 1H), 6.17 (dd, J=15.6, 8.0 Hz, 1H), 3.49 (d, J=7.2 Hz, 1H), 2.11(dd, J=13.2, 1.6 Hz, 3H), 2.02 (d, J=4.8 Hz, 3H), 1.24-1.88 (m, 3H). ¹⁹FNMR (376 MHz, DMSO-D₆) δ ppm −115.68. LCMS (ESI+) m/z: [MH]⁺ calcd forC₁₄H₁₇FNO₃+: 266.1, found: 266.1.

4-(5-Acetamido-3-fluoro-2-methylphenyl)-2-methylbutanoic acid (8-62)

To a mixture of4-(5-acetamido-3-fluoro-2-methylphenyl)-2-methylbut-3-enoic acid (1)(5.00 g, 18.8 mmol, 1.0 equiv) in methanol (20 mL) was added Pd/C (10 wt%) (2.40 g, 0.12 equiv) under nitrogen, the suspension was degassedunder vacuum and purged with H₂ three times, and stirred under H₂ (15psi) at 25° C. for 10 h. After the H₂ atmosphere was replaced withargon, it was filtered through a pad of Celite and filter cake washedwith methanol (300 mL). The combined filtrates were concentrated underreduced pressure to give4-(5-acetamido-3-fluoro-2-methylphenyl)-2-methylbutanoic acid (8-62)(4.4 g, 87% yield). ¹H NMR (400 MHz, DMSO-D₆) δ ppm 10.0 (s, 1H), 7.42(dd, J=12.4, 1.6 Hz, 1H), 7.04 (s, 1H), 2.52-2.58 (m, 2H), 2.24 (t,J=6.8 Hz, 2H), 2.08 (d, J=1.6 Hz, 3H), 2.01 (s, 3H), 1.40-1.65 (m, 4H).¹⁹F NMR (376 MHz, DMSO-D₆) δ ppm −125.77. LCMS (ESI+) m/z: [MH]⁺ calcdfor C₁₄H₁₉FNO₃ ⁺: 268.1, found: 268.1.

N-(7-Fluoro-3,8-dimethyl-4-oxotetralin-5-yl)acetamide (8-63)

To a mixture of 4-(5-acetamido-3-fluoro-2-methylphenyl)-2-methylbutanoicacid (8-62) (5.00 g, 18.7 mmol, 1.0 equiv) in trifluoroacetic acid (10mL) was added trifluoroacetic anhydride (5.20 mL, 37.4 mmol, 2.0 equiv)at 0° C., the mixture was stirred at 0° C. for 2 h, quenched with icewater (100 mL), and the pH adjusted to around 7 by addition of 25%aqueous NaOH at 0° C. It was extracted with ethyl acetate (3×200 mL),the combined organic layers washed with brine, dried over Na₂SO₄,filtered, concentrated under reduced pressure, and the residue purifiedby silica gel flash column chromatography eluting with 8% ethyl acetatein petroleum ether to giveN-(7-fluoro-3,8-dimethyl-4-oxotetralin-5-yl)acetamide (8-63) (3.50 g,75% yield). ¹H NMR (400 MHz, DMSO-D₆) δ ppm 12.13 (s, 1H), 8.28 (d,J=13.2 Hz, 1H), 2.93-3.04 (m, 1H), 2.81-2.92 (m, 1H), 2.63-2.75 (m, 1H),2.07-2.18 (m, 7H), 1.74 (qd, J=12.0, 4.8 Hz, 1H), 1.15 (d, J=6.4 Hz,3H). ¹⁹F NMR (376 MHz, DMSO-D₆) δ ppm −104.64. LCMS (ESI+) m/z: [MH]⁺calcd for C₁₄H₁₇FNO₂ ⁺: 250.1, found: 250.1.

Di-tert-Butyl1-(8-acetamido-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)hydrazine-1,2-dicarboxylate(8-64)

To a mixture of N-(7-fluoro-3,8-dimethyl-4-oxotetralin-5-yl)acetamide(8-63) (35.1 g, 140 mmol, 1.0 equiv) in toluene (700 mL) was addedsodium bis(trimethylsilyl)amide (309 mL, 1 M, 2.2 equiv) dropwise at 0°C. under nitrogen, the mixture was cooled down to −40° C., a solution ofdi-tert-butyl diazene-1,2-dicarboxylate (42.1 g, 183 mmol, 1.3 equiv) intoluene (350 mL) was added dropwise. The reaction mixture was warmed upto 25° C., stirred at 25° C. for 4 h, cooled to 0° C., diluted withwater (1 L) and extracted with ethyl acetate (3×500 mL). The combinedorganic layers were washed with brine, dried over Na₂SO₄, filtered,concentrated under reduced pressure, and the residue purified by silicagel flash column chromatography eluting with 20% of ethyl acetate inpetroleum ether to give di-tert-butyl1-(8-acetamido-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)hydrazine-1,2-dicarboxylate (8-64) (41.0 g, 60% yield). ¹H NMR (400 MHz,DMSO-D₆) δ ppm 11.61-11.84 (m, 1H), 8.26 (d, J=12.8 Hz, 1H), 7.96-8.15(m, 1H), 2.96-3.16 (m, 2H), 2.68-2.84 (m, 1H), 2.12 (s, 4H), 2.08 (s,3H), 1.35-1.46 (m, 21H). ¹⁹F NMR (376 MHz, DMSO-D₆) δ ppm −105.29. LCMS(ESI+) m/z: [MH]⁺ calcd for C₂₄H₃₅FN₃O₆ ⁺: 480.2, found: 502 (MS+Na).

N-(3-Fluoro-4,7-dimethyl-8-oxo-7-(2-(propan-2-ylidene)hydrazinyl)-5,6,7,8-tetrahydrona-phthalen-1-yl)acetamide(8-66)

To a solution of di-tert-butyl1-(8-acetamido-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)hydrazine-1,2-dicarboxylate(8-64) (41.0 g, 85.5 mmol, 1.0 equiv) in dichloromethane (820 mL) wasadded trifluoroacetic acid (410 mL) at 25° C., the mixture was stirredat 25° C. for 1 h, acetone (480 mL) added and the mixture stirred at 25°C. for another 0.5 h. It was concentrated to giveN-(3-fluoro-4,7-dimethyl-8-oxo-7-(2-(propan-2-ylidene)hydrazinyl)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide(8-66) (18.1 g, 66% yield). ¹H NMR (400 MHz, DMSO-D₆) δ ppm 11.90 (s,1H), 8.31 (d, J=12.8 Hz, 1H), 3.05-3.14 (m, 1H), 2.90-3.02 (m, 1H),2.11-2.19 (m, 7H), 2.05-2.08 (m, 2H), 1.98 (d, J=2.0 Hz, 6H), 1.34 (s,3H). ¹⁹F NMR (376 MHz, DMSO-D₆) δ ppm −75.04. LCMS (ESI+) m/z: [MH]⁺calcd for C₁₇H₂₃FN₃O₂ ⁺: 320.1, found: 320.1.

N-(7-Amino-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide(8-67)

To a mixture ofN-(3-fluoro-4,7-dimethyl-8-oxo-7-(2-(propan-2-ylidene)hydrazinyl)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide(8-66) (15.1 g, 47.3 mmol, 1.0 equiv) in acetic acid (302 mL) was addedzinc powder (40.8 g, 624 mmol, 13.2 equiv) portion wise, the mixture wasstirred at 20° C. for 2 h, filtered and the filtrate concentrated underreduced pressure to giveN-(7-amino-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide(8-67) (23.8 g, crude), which was used directly in the next step withoutfurther purification. ¹H NMR (400 MHz, DMSO-D₆) δ ppm 11.56 (s, 1H),8.26-8.30 (m, 1H), 3.01-3.09 (m, 2H), 2.12-2.19 (m, 10H), 1.43 (s, 3H).¹⁹F NMR (376 MHz, DMSO-D₆) δ ppm −73.57. LCMS (ESI+) m/z: [MH]⁺ calcdfor C₁₄H₁₈FN₂O₂ ⁺: 265.1, found: 265.1.

N,N′-(3-Fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide(8-68)

To a mixture ofN-(7-amino-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide(8-67) (23.8 g, 73.3 mmol, 1.0 equiv) in dichloromethane (414 mL) wereadded acetic anhydride (8.28 mL, 88.0 mmol, 1.2 equiv) and triethylamine(30.6 mL, 220 mmol, 3.0 equiv), the mixture was stirred at 25° C. for 12h, quenched with water (500 mL) and extracted with dichloromethane(3×200 mL). The combined organic layers were washed with brine, driedover Na₂SO₄, filtered, concentrated under reduced pressure, and theresidue purified by silica gel flash column chromatography eluting with70% ethyl acetate in petroleum ether to giveN,N′-(3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide(8-68) (7.5 g, 51% yield). ¹H NMR (400 MHz, DMSO-D₆) δ ppm 11.81 (s,1H), 8.20-8.37 (m, 2H), 2.94-3.03 (m, 1H), 2.77-2.88 (m, 1H), 2.65 (m,1H), 2.06-2.18 (m, 6H), 1.85 (m, 1H), 1.79 (s, 3H), 1.32 (s, 3H). ¹⁹FNMR (376 MHz, DMSO-D₆) δ ppm −105.03.

N-(8-Amino-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide(8-69)

To a mixture ofN,N′-(3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide(8-68) (7.50 g, 24.4 mmol, 1.0 equiv) in methanol (105 mL) was addedHCl/MeOH (105 mL, 4 M), the mixture was stirred at 25° C. for 2 h,concentrated under reduced pressure, the residue diluted withdichloromethane (300 mL) and extracted with saturated aqueous NaHCO₃(2×200 mL). The organic layer was washed with brine, dried over Na₂SO₄,filtered, and concentrated under reduced pressure to giveN-(8-amino-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide(8-69) (5.50 g, 89% yield). ¹H NMR (400 MHz, DMSO-D₆) δ ppm 7.91 (s,1H), 7.39 (s, 2H), 6.36 (d, J=12.4 Hz, 1H), 2.78-2.89 (m, 1H), 2.67-2.75(m, 2H), 1.97 (d, J=1.20 Hz, 3H), 1.83-1.88 (m, 1H), 1.80 (s, 3H), 1.27(s, 3H). ¹⁹F NMR (376 MHz, DMSO-D₆) δ ppm −108.38. LCMS (ESI+) m/z:[MH]⁺ calcd for C₁₄H₁₈FN₂O₂ ⁺: 265.1, found: 265.1.

N-((9S)-9-Ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydro-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide(8-70)

To a mixture ofN-(8-amino-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide(500 mg, 1.89 mmol, 1.0 equiv) (8-69) and(S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (547 mg, 2.08 mmol, 1.1 equiv) in toluene (25 mL) at120° C. were added pyridine 4-methylbenzenesulfonate (71.3 mg, 0.283mmol, 0.15 equiv) and o-cresol (1.44 mL, 13.8 mmol, 7.3 equiv) underargon, and the mixture was stirred at 130° C. for 13 h with Dean-Starktrap to remove the water formed. It was concentrated under reducedpressure, and the residue purified by silica gel flash columnchromatography eluting with 7% methanol in dichloromethane to affordN-((9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]py-rano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide(8-70) (362 mg, 39% yield). ¹H NMR (400 MHz, DMSO-D₆) δ ppm 7.78 (d,J=11.2 Hz, 1H), 7.30 (s, 1H), 6.53 (d, J=10.0 Hz, 1 H), 5.25-5.54 (m,4H), 4.81-4.90 (m, 1H), 3.24-3.29 (m, 1H), 2.86-3.11 (m, 3H), 2.39 (s,3H), 1.95 (d, J=3.6 Hz, 3H), 1.84-1.89 (m, 2H), 1.50 (d, J=4.8 Hz, 3H),0.87 (d, J=5.2 Hz, 3H). ¹⁹F NMR (376 MHz, DMSO-D₆) δ ppm −111.94. LCMS(ESI+) m/z: [MH]⁺ calcd for C₂₇H₂₇FN₃O₅ ⁺: 492.1, found: 492.2

(1S,9S)-1-Amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]py-rano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (8-71) and(1R,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (8-72)

A mixture ofN-((9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)acet-amide(8-70)(600 mg, 1.22 mmol, 1.0 equiv) in dioxane (6 mL) and concentratedhydrochloric acid (6 mL, 12 M) was stirred at 100° C. for 24 h in asealed tube under argon. It was concentrated under reduced pressure, andthe residue purified by prep-HPLC to afford(1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H, 9H)-dione hydrochloride (8-71) (61.0 mg, 10% yield) and(1R,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indoli-zino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (8-72) (85.0 mg, 14% yield). Note: thestereochemistry is arbitrarily assigned.

Prep-HPLC Conditions:

-   -   Instrument: Gilson 281 semi-preparative HPLC system    -   Mobile phase: A: HCl/H2O=0.040% v/v; B: ACN    -   Column: Phenomenex Luna 80*30 mm*3 um    -   Flow rate: 25 mL/min    -   Monitor wavelength: 220&254 nm

Time B % 0.0 5 8.0 30 8.1 30 8.2 100 10.2 100 10.3 5 11.5 5

Spectra of 8-71: ¹H NMR (400 MHz, DMSO-D₆) δ ppm 8.97 (s, 3H), 7.89 (d,J=10.8 Hz, 1H), 7.36 (s, 1H), 5.73-5.81 (m, 1H), 5.56-5.65 (m, 1H),5.41-5.49 (m, 2H), 3.22 (d, J=4.4 Hz, 2H), 2.34-2.44 (m, 5H), 1.82-1.94(m, 5H), 0.88 (t, J=7.2 Hz, 3H). ¹⁹F NMR (376 MHz, DMSO-D₆) δ ppm−111.24. LCMS (ESI+) m/z: [MH]⁺ calcd for C₂₅H₂₅FN₃O₄ ⁺: 450.1, found:450.1

Spectra of 8-72: ¹H NMR (400 MHz, DMSO-D₆) δ ppm 8.98 (s, 3H), 7.89 (d,J=10.8 Hz, 1H), 7.36 (s, 1H), 5.71-5.79 (m, 1H), 5.57-5.64 (m, 1H), 5.46(s, 2H), 3.18-3.26 (m, 2H), 2.36-2.44 (m, 5H), 1.81-1.94 (m, 5H), 0.87(t, J=7.2 Hz, 3H). ¹⁹F NMR (376 MHz, DMSO-D₆) δ ppm −111.22. LCMS (ESI+)m/z: [MH]⁺ calcd for C₂₅H₂₅FN₃O₄ ⁺: 450.1, found: 450.1

Example 9N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide(9-74) (FIG. 20)

2-(((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octa-hydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethylacetate (9-73)

The mixture of(1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetra-hydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (8-71) (40.0 mg, 0.089 mmol, 1.0 equiv)),N-ethyl-N-isopropylpropan-2-amine (46.5 μL, 0.266 mmol, 3.0 equiv) inN,N-dimethylformamide (2 mL) was stirred at 25° C. for 0.5 h, cooled to0° C., 2-Chloro-2-oxoethyl acetate (14.6 mg, 11.5 μL, 0.106 mmol, 1.2equiv) added dropwise over 5 min. It was warmed up to 25° C. and stirredat 25° C. for 3 h, concentrated under reduced pressure and the residuepurified by silica gel flash column chromatography eluting withchloroform/methanol/water=7/3/1 to give2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethylacetate (9-73) (30.0 mg, 61% yield). ¹H NMR (400 MHz, DMSO-D₆) δ ppm8.84 (s, 1H), 7.78 (d, J 10.85 Hz, 1H), 7.30 (s, 1H), 6.51 (s, 1H),5.35-5.49 (m, 3H), 5.13 (d, J 19.07 Hz, 1H), 4.66 (d, J=14.66 Hz, 1H),4.50 (d, J=14.66 Hz, 1H), 2.93-3.09 (m, 3H), 2.39 (s, 3H), 1.99 (s, 3H),1.83-1.95 (m, 3H), 1.55 (s, 3H), 0.88 (t, J=7.33 Hz, 3H). ¹⁹F NMR (376MHz, DMSO-D₆) δ ppm −111.87. LCMS (ESI+) m/z: [MH]⁺ calcd forC₂₉H₂₉FN₃O₇ ⁺: 550.2, found: 550.2.

N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octa-hydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide(9-74)

To a mixture of2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethylacetate (9-73) (30.0 mg, 0.055 mmol, 1.0 equiv) in tetrahydrofuran (1mL) was added 1 N aqueous NaOH (0.235 mL, 4.3 equiv) at 25° C. underargon. The mixture was stirred at 25° C. for 2 h, quenched with 1 Naqueous hydrochloric acid (0.273 mL, 5.0 equiv), concentrated underreduced pressure, and the residue purified by silica gel flash columnchromatography eluting with chloroform/methanol/water=7/3/1) to giveN-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydro-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide(9-74) (13.5 mg, 50% yield). ¹H NMR (400 MHz, DMSO-D₆) δ ppm 8.31 (s,1H), 7.77 (d, J=10.85 Hz, 1H), 7.29 (s, 1H), 6.50 (s, 1H), 5.34-5.54 (m,4H), 4.91 (d, J=19.07 Hz, 1H), 3.77-4.01 (m, 2H), 3.22-3.31 (m, 1H),2.87-3.13 (m, 2H), 2.39 (s, 3H), 1.77-2.01 (m, 3H), 1.58 (s, 3H), 0.87(t, J=7.33 Hz, 3H). ¹⁹F NMR (376 MHz, DMSO-D₆) δ ppm −111.92. LCMS(ESI+) m/z: [MH]⁺ calcd for C₂₇H₂₇FN₃O₆ ⁺: 508.2, found: 508.1.

Example 10N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quino-lin-1-yl)-2-hydroxyacetamide(10-75)

Example 10 (10-75) was made in a similar fashion to Example 9 using 8-72instead of 8-71.

¹H NMR (400 MHz, DMSO-D₆) δ ppm 8.31 (s, 1H), 7.77 (d, J=10.85 Hz, 1H),7.29 (s, 1H), 6.50 (s, 1H), 5.34-5.54 (m, 4H), 4.91 (d, J=19.07 Hz, 1H),3.77-4.01 (m, 2H), 3.22-3.31 (m, 1H), 2.87-3.13 (m, 2H), 2.39 (s, 3H),1.77-2.01 (m, 3H), 1.58 (s, 3H), 0.87 (t, J=7.33 Hz, 3H). ¹⁹F NMR (376MHz, DMSO-D₆) δ ppm −111.88. LCMS (ESI+) m/z: [MH]⁺ calcd forC₂₇H₂₇FN₃O₆ ⁺: 508.2, found: 508.1.

Example 11N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-4-hydroxybutanamidehydrochloride (11-80) (FIG. 21)

4-((tert-butyldiphenylsilyl)oxy)butanoic acid (11-77)

To a mixture of sodium 4-hydroxybutanoate (11-76) (2.10 g, 16.6 mmol,1.0 equiv) and imidazole (1.70 g, 25.0 mmol, 1.5 equiv) in N,N-dimethylformamide (31.5 mL) was added tert-butylchlorodiphenylsilane(5.49 g, 19.9 mmol, 1.2 equiv) under argon, the mixture was stirred at25° C. for 1 h, poured into ice water (60 mL) and extracted with ethylacetate (3×50 mL). The combined organic layers were washed with brine,dried over Na₂SO₄, filtered, concentrated under reduced pressure, andthe residue purified by silica gel flash column chromatography elutingwith 30% ethyl acetate in petroleum ether to give4-((tert-butyldiphenylsilyl)oxy)butanoic acid (11-77) (2.60 g, 41%yield). ¹H NMR (400 MHz, DMSO-D₆) δ ppm 7.61-7.63 (m, 6H), 7.46 (dd,J=7.2, 4.4 Hz, 4H), 3.72 (t, J=6.4 Hz, 2H), 2.65 (t, J=7.2 Hz, 2H), 1.87(t, J=6.8 Hz, 2H), 1.00 (s, 9H). LCMS (ESI−) m/z: [MH]⁻ calcd forC₂₀H₂₅O₃Si⁻: 341.2, found: 341.2.

4-((tert-Butyldiphenylsilyl)oxy)butanoyl chloride (11-78)

To a mixture of 4-((tert-butyldiphenylsilyl)oxy)butanoic acid (11-77)(1.50 g, 4.10 mmol, 1.0 equiv) in dichloromethane (75 mL) were addedoxalyl dichloride (1.05 g, 8.24 mmol, 2.0 equiv) and N,N-dimethylformamide (9.02 mg, 0.03 equiv) under nitrogen at 0° C., themixture was warmed up to 25° C., stirred at 25° C. for 2 h, andconcentrated under reduced pressure to give4-((tert-butyldiphenylsilyl)oxy)butanoyl chloride (11-78) (1.50 g,crude), which was used directly in the next step without purification.¹H NMR (400 MHz, CDCl₃) δ ppm 7.63-7.71 (m, 6H), 7.42-7.48 (m, 4H), 3.72(dt, J=11.6, 6.0 Hz, 2H), 2.96-3.13 (m, 1H), 2.63 (t, J=7.6 Hz, 1H),1.91-2.01 (m, 2H), 1.07 (s, 9H).

4-((tert-Butyldiphenylsilyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)butanamide(11-79)

To a mixture of(1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetra-hydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (8-71) (100 mg, 0.206 mmol, 1.0 equiv) andtrimethylamine (166 mg, 229 L 1.65 mmol, 8.0 equiv) in dichloromethane(5 mL) was added 4-((tert-butyldiphenylsilyl) oxy)butanoyl chloride(11-78) (303 mg, 0.839 mmol, 2.4 equiv), the mixture was stirred at 25°C. for 1 h, filtered, the filtrate concentrated and the residue purifiedby silica gel flash column chromatography eluting with 60%dichloromethane in ethyl acetate to give4-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)butanamide(11-79) (30.0 mg, 17% yield). LCMS (ESI+) m/z: [MH]⁺ calcd forC₄₅H₄₉FN₃O₆Si⁺: 774.3, found: 774.2.

N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-4-hydroxybutanamidehydrochloride (11-80)

To a stirring mixture of4-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)butanamide(11-79) (30.0 mg, 0.039 mmol, 1.0 equiv) in dioxane (0.15 mL) was addedHCl/dioxane (0.5 mL, 8 M) dropwise at 25° C. under nitrogen, the mixturewas stirred at 25° C. for 1 h, filtered, and the filter cake collected.The material was triturated with methyl tert-butyl ether (2 mL) at 25°C. for 30 min, collected by filtration, and dried under vacuum to giveN-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-4-hydroxybutanamidehydrochloride (11-80) (10.2 mg, 46% yield). ¹H NMR (400 MHz, DMSO-D₆) δppm 8.61 (s, 1H), 7.78 (d, J 10.8 Hz, 1H), 7.31 (s, 1H), 5.35-5.48 (m,3H), 4.87 (d, J=18.8 Hz, 1H), 3.37 (t, J=6.4 Hz, 3H), 3.22-3.32 (m, 1H),2.86-3.11 (m, 3H), 2.39 (s, 3H), 2.25-2.33 (m, 2H), 1.79-1.95 (m, 3H),1.56-1.66 (m, 2H), 1.51 (s, 3H), 0.88 (t, J=7.2 Hz, 3H). ¹⁹F NMR (376MHz, DMSO-D₆) δ ppm −111.98. LCMS (ESI+) m/z: [MH]⁺ calcd forC₂₉H₃₁FN₃O₆ ⁺: 536.2, found: 536.2.

Example 12N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-4-hydroxybutanamidehydrochloride (12-81)

Example 12 (12-81) was made in a similar fashion to Example 11 using8-72 instead of 8-71. ¹H NMR (400 MHz, DMSO-D6) δ ppm 8.60 (s, 1H), 7.78(d, J=10.8 Hz, 1H), 7.30 (s, 1H), 5.31-5.50 (m, 3H), 4.86 (d, J=18.8 Hz,1H), 3.36 (t, J=6.4 Hz, 2H), 3.23-3.31 (m, 1H), 2.78-3.15 (m, 3H), 2.39(s, 3H), 2.26-2.34 (m, 2H), 1.75-1.99 (m, 3H), 1.60 (quin, J=6.8 Hz,2H), 1.50 (s, 3H), 0.86 (t, J=7.2 Hz, 3H). ¹⁹F NMR (376 MHz, DMSO-D6) δppm −111.95. LCMS (ESI+) m/z: [MH]⁺ calcd for C₂₉H₃₁FN₃O₆ ⁺: 536.2,found: 536.1.

Example 13(1S,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13 (1H,9H)-dione (13-84) and(1R,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13 (1H,9H)-dione (13-85) (FIG. 22)

N-(3-Fluoro-7-hydroxy-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide(13-82)

To a mixture ofN-(3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide(8-63) (1.00 g, 4.01 mmol, 1.0 equiv) and cesium carbonate (261 mg,0.802 mmol, 0.2 equiv) in dimethyl sulfoxide (16 mL) was added triethylphosphite (1.33 g, 8.02 mmol, 1.38 mL, 2.0 equiv) at 25° C., thereaction mixture was vacuumed and filled with 02 three times and stirredunder 02 (15 psi) at 25° C. for 24 h. After the oxygen atmosphere wasreplaced with nitrogen, the mixture was diluted with water (80 mL) andextracted with ethyl acetate (2×40 mL). The combined organic layers werewashed with brine, dried over Na₂SO₄, filtered, concentrated underreduced pressure, and the residue purified by silica gel flash columnchromatography eluting with 15% of ethyl acetate in petroleum ether toaffordN-(3-fluoro-7-hydroxy-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide(12-82) (620 mg, 58% yield). ¹H NMR (400 MHz, DMSO-D₆) δ ppm 11.97 (s,1H), 8.29 (d, J=13.08 Hz, 1H), 5.47 (s, 1H), 2.94-3.04 (m, 1H),2.78-2.88 (m, 1H), 2.17 (s, 3H), 2.11 (d, J=1.59 Hz, 3H), 1.96-2.09 (m,2H), 1.28 (s, 3H). ¹⁹F NMR (376 MHz, DMSO-D₆) δ ppm −104.38. LCMS (ESI+)m/z: [MH]⁺ calcd for C₁₄H₁₇FNO₃ ⁺: 266.1, found: 266.0.

8-Amino-6-fluoro-2-hydroxy-2,5-dimethyl-3,4-dihydronaphthalen-1 (2H)-one(12-83)

To a mixture ofN-(3-fluoro-7-hydroxy-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide(12-82) (300 mg, 1.13 mmol, 1.0 equiv) in methanol (6 mL) was addedHCl/MeOH (6 mL, 4 M) dropwise at 25° C., the mixture was stirred at 25°C. for 1.5 h, cooled to 0° C. and the pH adjusted to 7 by addition ofsaturated NaHCO₃ at 0° C. It was extracted with dichloromethane (3×10mL), the combined organic layers dried over Na₂SO₄, filtered,concentrated under reduced pressure to give8-amino-6-fluoro-2-hydroxy-2,5-dimethyl-3,4-dihydronaphthalen-1 (2H)-one(13-83) (250 mg, 99% yield), which was used directly in the next stepwithout further purification. ¹H NMR (400 MHz, DMSO-D₆) δ ppm 7.41 (brs, 2H), 6.37 (d, J=12.59 Hz, 1H), 5.10 (s, 1H), 2.81-2.92 (m, 1H),2.63-2.75 (m, 1H), 1.89-2.01 (m, 5H), 1.22 (s, 3H). ¹⁹F NMR (376 MHz,DMSO-D₆) δ ppm −108.05.

(1S,9S)-9-Ethyl-5-fluoro-1,9-dihydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13 (1H,9H)-dione (13-84) and(1R,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13 (1H,9H)-dione (13-85)

To a mixture of8-amino-6-fluoro-2-hydroxy-2,5-dimethyl-3,4-dihydronaphthalen-1 (2H)-one(13-83) (54.0 mg, 241 umol, 1.0 equiv) and(S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (95.5 mg, 0.362 mmol, 1.5 equiv) in xylene (5.4 mL)was added 4-methylbenzenesulfonic acid (24.9 mg, 0.145 mmol, 0.6 equiv)at 120° C. under argon, the mixture was stirred at 120° C. for 24 h in asealed tube. It was concentrated under reduced pressure, and the residuepurified by silica gel flash column chromatography eluting with 60% oftetrahydrofuran in petroleum ether to give a residue, which was furtherseparated by prep-HPLC and lyophilization to afford(1S,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (13-84) (70.1 mg, 13% yield) (compound 13-84 may be theopposite enantiomer of that depicted), and(1R,9S)-9-ethyl-5-fluoro-1,9-dihydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (13-85) (73.3 mg, 13% yield) (compound 13-85 may be theopposite enantiomer of that depicted). Note: the stereochemistry isarbitrarily assigned.

Prep-HPLC Method:

-   -   Instrument: Gilson 281 semi-preparative HPLC system    -   Mobile phase: A: H₂O; B: CH₃OH    -   Column: Phenomenex Luna 80*30 mm*3 um    -   Flow rate: 25 mL/min    -   Monitor wavelength: 220&254 nm

Time B % 0.0 40 8.0 70 8.1 70 8.2 100 10.2 100 10.3 40 11.5 40

Spectra of 13-84: ¹H NMR (400 MHz, DMSO-D₆) δ ppm 7.76 (d, J=10.88 Hz,1H), 7.31 (s, 1H), 6.51 (s, 1H), 5.79 (br s, 1H), 5.43 (s, 4H), 3.25 (brd, J=2.57 Hz, 1H), 2.96-3.09 (m, 1H), 2.38 (s, 3H), 2.24 (br dd,J=13.08, 3.06 Hz, 1H), 2.11 (td, J=13.24, 5.69 Hz, 1H), 1.87 (m, 2H),1.45 (s, 3H), 0.88 (t, J=7.34 Hz, 3H). ¹⁹F NMR (376 MHz, DMSO-D₆) δ ppm−112.10. LCMS (ESI+) m/z: [MH]⁺ calcd for C₂₅H₂₄FN₂O₅ ⁺: 451.1, found:451.1. SFC: RT=1.338 min.

Spectra of 13-85: ¹H NMR (400 MHz, DMSO-D₆) δ ppm 7.77 (d, J=11.00 Hz,1H), 7.31 (s, 1H), 6.50 (s, 1H), 5.80 (br s, 1H), 5.44 (s, 4H), 3.25 (brs, 1H), 2.96-3.10 (m, 1H), 2.38 (s, 3H), 2.24 (br dd, J=12.65, 3.24 Hz,1H), 2.12 (td, J=13.14, 5.38 Hz, 1H), 1.78-1.93 (m, 2H), 1.44 (s, 3H),0.87 (t, J=7.27 Hz, 3H). ¹⁹F NMR (376 MHz, DMSO-D₆) δ ppm −112.07. LCMS(ESI+) m/z: [MH]⁺ calcd for C₂₅H₂₄FN₂O₅ ⁺: 451.1, found: 451.1. SFC:RT=1.453 min.

Example 14(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indo-lizino[1,2-b]quinoline-10,13(1H,9H)-dione (14-91) and(1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (14-92) (FIG. 23)

N-(7-(Allyloxy)-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (14-86)

To a mixture ofN-(3-fluoro-7-hydroxy-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydro-naphthalen-1-yl)acetamide(13-82) (500 mg, 1.88 mmol, 1.0 equiv) and Ag₂O (4.37 g, 18.8 mmol, 10equiv) in acetonitrile (10 mL) was added 3-iodoprop-1-ene (6.33 g, 3.44mL, 37.7 mmol, 20 equiv) at 20° C. under argon, and the mixture wasstirred at 40° C. for 12 h and cooled to 25° C. (Three additionalreactions were set up as described above and four reaction mixtures werecombined). The combined reaction mixtures were filtered through a pad ofCelite, and the filter cake washed with dichloromethane (200 mL),combined filtrates concentrated, and the residue purified by silica gelflash column chromatography eluting with 5% ethyl acetate in petroleumether to giveN-(7-(allyloxy)-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide(14-86) (1.30 g, 54% yield). ¹H NMR (400 MHz, DMSO-D₆) δ ppm 11.82 (s,1H), 8.28 (d, J=13.13 Hz, 1H), 5.70-5.83 (m, 1H), 5.14 (m, 1H), 5.01 (m,1H), 3.99 (m, 1H), 3.79 (m, 1H), 3.00 (m, 1H), 2.77-2.88 (m, 1H), 2.36(dt, J=14.07, 5.22 Hz, 1H), 2.17 (s, 3H), 2.11 (d, J=1.63 Hz, 3H), 2.03(m, 1H), 1.30-1.38 (m, 3H). ¹⁹F NMR (376 MHz, DMSO-D₆) δ ppm −104.04.LCMS (ESI+) m/z: [MH]⁺ calcd for C₁₇H₂₁FNO₃ ⁺: 306.1, found: 306.1.

N-(3-Fluoro-4,7-dimethyl-8-oxo-7-(2-oxoethoxy)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide(14-87)

To a stirred mixture ofN-(7-(allyloxy)-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydro-naphthalen-1-yl)acetamide(14-86) (1.30 g, 4.17 mmol, 1.0 equiv) in dichloromethane (26 mL) andmethanol (13 mL) at −78° C., was bubbled ozone over 5 min, followed byaddition of dimethylsulfane (648 mg, 0.766 mL, 10.4 mmol, 2.5 equiv),the reaction mixture was warmed up to 25° C. and stirred at 25° C. for 1h, quenched with water (40 mL) and extracted with dichloromethane (3×40mL). The combined organic layers were washed with brine, dried overNa₂SO₄, filtered, concentrated under reduced pressure to giveN-(3-fluoro-4,7-dimethyl-8-oxo-7-(2-oxoethoxy)-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide(14-87) (1.20 g, crude), which was used directly in the next reactionwithout further purification.

N-(3-Fluoro-7-(2-hydroxyethoxy)-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphtha-len-1-yl)acetamide(14-88)

To a mixture ofN-(3-fluoro-4,7-dimethyl-8-oxo-7-(2-oxoethoxy)-5,6,7,8-tetrahydro-naphthalen-1-yl)acetamide(14-87) (1.20 g, 2.73 mmol, 1.0 equiv) in tetrahydrofuran (30 mL) andH₂O (15 mL) at 0° C. was added sodium tetrahydroboride (51.7 mg, 1.37mmol, 0.5 equiv) portion wise, the mixture was stirred at 0° C. for 10min, warmed up to 25° C. and stirred at 25° C. for 20 min, quenched bywater (30 mL) and extracted with dichloromethane (3×40 mL). The combinedorganic layers were washed with brine, dried over Na₂SO₄, filtered,concentrated under reduced pressure, and the residue purified by silicagel flash column chromatography eluting with 40% ethyl acetate inpetroleum ether to giveN-(3-fluoro-7-(2-hydroxyethoxy)-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide(14-88) (800 mg, 61% yield). ¹H NMR (400 MHz, CDCl₃) δ ppm 12.07 (br s,1H), 8.46 (d, J=12.76 Hz, 1H), 3.63-3.76 (m, 2H), 3.53-3.61 (m, 1H),3.44-3.52 (m, 1H), 3.03-3.23 (m, 1H), 2.74-2.92 (m, 1H), 2.35-2.46 (m,1H), 2.22-2.26 (m, 3H), 2.12 (br s, 3H), 2.03-2.11 (m, 1H), 1.37-1.48(m, 3H). ¹⁹F NMR (376 MHz, CDCl₃) δ ppm −101.12. LCMS (ESI+) m/z: [MH]⁺calcd for C₁₆H₂₁FNO₄ ⁺: 310.1, found: 310.1.

8-Amino-6-fluoro-2-(2-hydroxyethoxy)-2,5-dimethyl-3,4-dihydronaphthalen-1(2H)-one (14-89)

To a solution ofN-(3-fluoro-7-(2-hydroxyethoxy)-4,7-dimethyl-8-oxo-5,6,7,8-tetra-hydronaphthalen-1-yl)acetamide(14-88) (200 mg, 581 umol, 1.0 equiv) in methanol (8 mL) under argon wasadded 2 N hydrochloric acid (8 mL) at 20° C., and the mixture wasstirred at 60° C. for 1 h, and cooled to 0° C. (Three additionalreactions were set up as described above and four reaction mixtures werecombined). The combined reaction mixtures were cooled to 0° C., pHadjusted to 8 by addition of saturated NaHCO₃, warmed up to 25° C., andextracted with dichloromethane (2×100 mL). The combined organic layerswashed with brine, dried over Na₂SO₄, filtered, concentrated underreduced pressure, and the residue purified by silica gel flash columnchromatography eluting with 40% ethyl acetate in petroleum ether to give8-amino-6-fluoro-2-(2-hydroxyethoxy)-2,5-dimethyl-3,4-dihydronaphthalen-1(2H)-one (14-89) (310 mg, 45% yield). ¹H NMR (400 MHz, CD₃OD) δ ppm 6.30(d, J 12.26 Hz, 1H), 3.49-3.61 (m, 3H), 3.39 (m, 1H), 3.01-3.10 (m, 1H),2.69-2.79 (m, 1H), 2.32 (m, 1H), 2.00 (d, J=2.25 Hz, 4H), 1.37 (s, 3H).¹⁹F NMR (376 MHz, CD₃OD) δ ppm −108.91. LCMS (ESI+) m/z: [MH]⁺ calcd forC₁₄H₁₉FNO₃ ⁺: 268.1, found: 268.1.

(9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (14-90)

To a mixture of8-amino-6-fluoro-2-(2-hydroxyethoxy)-2,5-dimethyl-3,4-dihydro-naphthalen-1(2H)-one (14-89) (150 mg, 0.561 mmol, 1.0 equiv) and(S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1-13) (162 mg, 0.617 mmol, 1.1 equiv) in toluene (7 mL) at120° C. were added o-cresol (443 mg, 0.426 mL, 4.10 mmol, 7.3 equiv) andpyridine 4-methylbenzenesulfonate (21.1 mg, 0.084 mmol, 0.15 equiv)under argon, the mixture was stirred at 120° C. for 32 h in a seal tubeand cooled to 25° C. (One additional reaction was set up as describedabove and two reaction mixtures were combined). The combined reactionmixtures were concentrated under reduced pressure, and the residuepurified by silica gel flash column chromatography eluting with ethylacetate to give(9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H, 9H)-dione (14-90) (210 mg, 22% yield). ¹H NMR (400 MHz, DMSO-D₆) δppm 7.76 (dd, J=10.85, 3.46 Hz, 1H), 7.30 (s, 1H), 6.51 (d, J=2.15 Hz,1H), 5.32-5.62 (m, 4H), 4.74 (q, J=5.21 Hz, 1H), 3.58-3.71 (m, 3H),2.92-3.06 (m, 1H), 2.14-2.42 (m, 6H), 1.81-1.91 (m, 2H), 1.53 (br d,J=6.79 Hz, 3H), 0.81-0.94 (m, 3H). ¹⁹F NMR (376 MHz, DMSO-D₆) δ ppm−111.89. LCMS (ESI+) m/z: [MH]⁺ calcd for C₂₇H₂₈FN₂O₆ ⁺: 495.2, found:495.2.

(1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (14-91) and(1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxy-ethoxy)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (14-92)

(9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-1,4-dimethyl-2,3,12,15-tetra-hydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (14-90) (210 mg, 0.424 mmol) was dissolved in methanol andseparated by chiral SFC (Instrument: Waters SFC150 preparative SFC.Column: DAICEL CHIRALPAK AD(250 mm*30 mm, 10 um); Mobile phase: A forCO₂ and B for ethanol; Gradient: B %=50% isocratic elution mode; Flowrate: 70 g/min; Wavelength: 220 nm; Column temperature: 40° C.; Systemback pressure: 120 bar) to afford(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indo-lizino[1,2-b]quinoline-10,13(1H,9H)-dione (14-91) (65.1 mg, 31% yield) (compound 14-91 may be theopposite enantiomer of that depicted), and(1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (14-92) (62.5 mg, 30% yield) (compound 14-92 may be theopposite enantiomer of that depicted). Note: the stereochemistry isarbitrarily assigned.

Spectra of 14-91: ¹H NMR (400 MHz, DMSO-D₆) δ ppm 7.77 (br d, J=10.88Hz, 1H), 7.30 (s, 1H), 5.30-5.63 (m, 4H), 3.64 (br s, 3H), 3.28-3.31,(m, 2H), 2.99 (br s, 1H), 2.38 (br s, 3H), 2.33 (br s, 1H), 2.20 (br s,1H), 1.76-1.93 (m, 2H), 1.53 (br s, 3H), 0.81-0.92 (m, 3H). ¹⁹F NMR (376MHz, DMSO-D₆) δ ppm −111.87. LCMS (ESI+) m/z: [MH]⁺ calcd forC₂₇H₂₈FN₂O₆ ⁺: 495.2, found: 495.1. Chiral SFC: RT=1.715 min.

Spectra of 14-92: ¹H NMR (400 MHz, DMSO-D₆) δ ppm 7.77 (br d, J=10.88Hz, 1H), 7.31 (s, 1H), 5.33-5.57 (m, 4H), 3.65 (br s, 3H), 3.28-3.30,(m, 2H), 2.95 (br s, 1H), 2.38 (br s, 3H), 2.33 (br s, 1H), 2.17 (br s,1H), 1.76-1.93 (m, 2H), 1.53 (br s, 3H), 0.84-0.92 (m, 3H). ¹⁹F NMR (376MHz, DMSO-D₆) δ ppm −111.90. LCMS (ESI+) m/z: [MH]⁺ calcd forC₂₇H₂₈FN₂O₆ ⁺: 495.2, found: 495.3. Chiral SFC: RT=1.948 min.

Example 15(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethyl)amino)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (15-94) (FIG. 24)

(1S,9S)-1-((2-((tert-Butyldimethylsilyl)oxy)ethyl)amino)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (15-93)

A mixture of(1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydro-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione methanesulfonate (Exatecan) (100 mg, 0.188 mmol, 1.0equiv) and triethylamine (19.0 mg, 0.188 mmol, 1.0 equiv) in methanol (2mL) was stirred at 25° C. for 0.5 h, followed by addition of2-((tert-butyldimethylsilyl)oxy)acetaldehyde (32.8 mg, 0.188 mmol, 1.0equiv). After the reaction mixture was stirred at 25° C. for 1 h, sodiumcyanoborohydride (17.7 mg, 0.282 mmol, 1.5 equiv) added, and stirred at25° C. for additional 2 h. It was quenched by addition of water (0.2mL), diluted with dichloromethane (5 mL), dried over Na₂SO₄, filtered,concentrated under reduced pressure, and the residue purified byprep-HPLC to give(1S,9S)-1-((2-((tert-butyldimethyl-silyl)oxy)ethyl)amino)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (15-93) (25.0 mg, 22% yield). ¹H NMR (400 MHz, DMSO-D₆) δppm 7.74 (d, J=11.13 Hz, 1H), 7.30 (s, 1H), 6.51 (s, 1H), 5.34-5.47 (m,4H), 4.30 (br s, 1H), 3.69 (t, J=5.93 Hz, 2H), 3.15-3.24 (m, 1H),2.96-3.07 (m, 1H), 2.87 (br d, J=5.14 Hz, 1H), 2.73-2.82 (m, 1H), 2.37(s, 3H), 2.22 (dd, J=13.57, 4.89 Hz, 1H), 1.99-2.14 (m, 2H), 1.87 (dt,J=17.36, 7.09 Hz, 2H), 0.87 (t, J=7.34 Hz, 3H), 0.82 (s, 9H), 0.03 (d,J=3.18 Hz, 6H). ¹⁹F NMR (400 MHz, DMSO-D₆) δ ppm −111.83. LCMS (ESI+)m/z: [MH]⁺ calcd for C₃₂H₄₁FN₃O₅Si⁺: 594.2, found: 594.2.

HPLC Method:

-   -   Instrument: Gilson 281 semi-preparative HPLC system    -   Mobile phase: A: 10 mM NH₄HCO₃ in H₂O; B: CH₃CN    -   Column: Waters Xbridge BEH C18 100*30 mm*10 um    -   Flow rate: 25 mL/min    -   Monitor wavelength: 220&254 nm

Time B % 0.0 65 10.0 95 10.1 95 10.2 100 12.2 100 12.3 65 13.5 65

(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethyl)amino)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (15-94)

To a mixture of(1S,9S)-1-((2-((tert-butyldimethylsilyl)oxy)ethyl)amino)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (15-93) (25 mg, 0.042 mmol, 1.0 equiv) in methanol (0.5mL) was added HCl/MeOH (2.5 mL, 4 M) dropwise at 25° C. under nitrogen,the mixture was stirred at 25° C. for 1 h, filtered, collected and driedunder vacuum to give(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethyl)amino)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (15-94) (18.0 mg, 87% yield). ¹H NMR (400MHz, DMSO-D₆) δ ppm 8.81-9.14 (m, 2H), 7.89 (d, J=10.85 Hz, 1H), 7.35(s, 1H), 6.56 (s, 1H), 5.54-5.67 (m, 1H), 5.37-5.50 (m, 3H), 5.28 (br d,J=0.95 Hz, 1H), 5.10 (br s, 1H), 3.71 (br s, 2H), 3.09-3.26 (m, 3H),2.79 (br d, J=13.95 Hz, 1H), 2.41 (s, 3H), 2.09-2.25 (m, 1H), 1.77-1.96(m, 2H), 0.88 (t, J=7.33 Hz, 3H). ¹⁹F NMR (376 MHz, DMSO-D₆) δ ppm−110.92. LCMS (ESI+) m/z: [MH]⁺ calcd for C₂₆H₂₇FN₃O₅ ⁺: 480.2, found:480.2.

Example 16(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethyl)amino)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (16-95)

Example 16 (16-95) was made in a similar fashion to Example 15 using8-71 instead of exatecan. ¹H NMR (400 MHz, CD₃OD) δ ppm 7.82 (d, J=10.49Hz, 1H), 7.68 (s, 1H), 5.71-5.78 (m, 1H), 5.57-5.65 (m, 2H), 5.41 (d,J=16.33 Hz, 1H), 3.74-3.84 (m, 2H), 3.35 (br s, 2H), 3.20-3.28 (m, 2H),2.74 (dt, J=14.16, 5.80 Hz, 1H), 2.39-2.53 (m, 4H), 2.09 (s, 3H), 1.98(qd, J=7.17, 3.99 Hz, 2H), 1.02 (t, J=7.33 Hz, 3H). ¹⁹F NMR (376 MHz,CD₃OD) δ ppm −111.87. LCMS (ESI+) m/z: [MH]⁺ calcd for C₂₇H₂₉FN₃O₅:494.2, found: 494.1.

Example 17(1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxyethyl)amino)-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (17-96)

Example 17 (17-96) was made in a similar fashion to Example 15 using8-72 instead of exatecan. ¹H NMR (400 MHz, CD₃OD) δ ppm 7.82 (d, J=10.39Hz, 1H), 7.68 (s, 1H), 5.73-5.81 (m, 1H), 5.63 (d, J=3.30 Hz, 1H), 5.58(s, 1H), 5.42 (d, J=16.26 Hz, 1H), 3.74-3.87 (m, 2H), 3.34 (br s, 2H),3.22-3.28 (m, 2H), 2.70-2.82 (m, 1H), 2.36-2.56 (m, 4H), 2.10 (s, 3H),1.89-2.04 (m, 2H), 1.01 (t, J=7.34 Hz, 3H). ¹⁹F NMR (376 MHz, CD₃OD) δppm −111.90. LCMS (ESI+) m/z: [MH]⁺ calcd for C₂₇H₂₉FN₃O₅: 494.2, found:494.1.

Example 18(S)-2-(2-(2-(3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanamido)acetamido)acetamido)-N-(2-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide(18-112) (FIG. 25)

2,5-Dioxopyrrolidin-1-yl2-(2-(((benzyloxy)carbonyl)amino)acetamido)acetate (18-98)

To a mixture of 2-[[2-(benzyloxycarbonylamino)acetyl]amino]acetic acid(18-97) (55.0 g, 206 mmol, 1.0 equiv) in acetonitrile (550 mL) wereadded 1-hydroxypyrrolidine-2,5-dione (26.1 g, 227 mmol, 1.1 equiv) andN¹-((ethylimino)methylene)-N³,N³-dimethylpropane-1,3-diaminehydrochloride (47.5 g, 247 mmol, 1.2 equiv), the mixture was stirred at25° C. for 4 h, cooled to −10° C. and white material formed. Thesuspension was diluted with water (50 mL), stirred, filtered, and thefilter cake dried under vacuum to give 2,5-dioxopyrrolidin-1-yl2-(2-(((benzyloxy)carbonyl)amino)acetamido)acetate (18-98) (85.0 g, 175mmol, 85% yield), which was used directly in next step withoutpurification. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 2.81 (s, 4H), 2.93-3.12(m, 2H), 3.69 (d, J=6.11 Hz, 2H), 4.27 (d, J=5.87 Hz, 2H), 5.04 (s, 2H),7.24-7.45 (m, 5H), 7.57 (t, J=6.11 Hz, 1H), 8.58 (t, J=5.87 Hz, 1H).LCMS (ESI+) m/z: [MH]⁺ calcd for C₁₆H₁₈N₃O₇ ⁺: 364.1, found: 364.1.

(S)-11-Benzyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecan-12-oicacid (18-99)

To a mixture of 2,5-dioxopyrrolidin-1-yl2-(2-(((benzyloxy)carbonyl)amino)acetamido)acetate (18-98) (85.0 g, 175mmol, 1.0 equiv) in acetonitrile (425 mL) and H₂O (425 mL) were added(2S)-2-amino-3-phenyl-propanoic acid (34.7 g, 210 mmol, 1.2 equiv) andtrimethylamine (26.8 mL, 193 mmol, 1.1 equiv), the mixture was stirredat 25° C. for 2 h, cooled to 0° C., hydrochloric acid (12 M, 16.2 mL,1.1 equiv) added and stirred at 0° C. for 6 h. The resulting suspensionwas warmed up to 25° C., filtered and the filter cake dried under vacuumto give(S)-11-benzyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecan-12-oicacid (18-99) (50.0 g, 108 mmol, 62% yield), which was used directly innext step without further purification. ¹H NMR (400 MHz, DMSO-d₆) δ ppm2.59 (s, 1H), 2.88 (dd, J=13.77, 9.00 Hz, 1H), 3.04-3.11 (m, 1H),3.60-3.79 (m, 4H), 4.43 (td, J=8.43, 5.19 Hz, 1H), 5.03 (s, 2H),7.15-7.40 (m, 10H), 7.50 (t, J=6.02 Hz, 1H), 8.04 (br t, J=5.60 Hz, 1H),8.16 (d, J=8.11 Hz, 1H), 12.72 (br d, J=5.01 Hz, 1H). LCMS (ESI+) m/z:[MH]⁺ calcd for C₂₁H₂₄N₃O₆ ⁺: 414.2, found: 414.2.

(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate(18-101)

To a mixture of2-[[2-(9H-fluoren-9-ylmethoxycarbonylamino)acetyl]amino]acetic acid(18-100) (100 g, 282 mmol, 1.0 equiv) in tetrahydrofuran (1.5 L) andacetic acid (300 mL) was added lead tetraacetate (150 g, 338 mmol, 1.2equiv), the mixture was stirred at 50° C. for 36 h, cooled to 25° C.,and filtered. The filtrate was washed with aqueous trisodium citratedihydrate solution (2.5 L, 20% wt) twice, organic phase concentrated toabout 2.0 L, water (2.5 L) added, and the mixture stirred at 5° C. for 2h. The formed precipitates were filtered, washed with a mixture of cold(5° C.) tetrahydrofuran and water (3:10, 1.2 L), and dried under vacuumto give (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methylacetate (18-101) (100 g, 86% yield). ¹H NMR (400 MHz, CDCl₃) δ ppm 2.09(s, 3H), 3.90 (d, J=5.01 Hz, 2H), 4.19-4.28 (m, 1H), 4.41-4.52 (m, 2H),5.25 (d, J=6.97 Hz, 2H), 5.41-5.60 (m, 1H), 7.17-7.27 (m, 1H), 7.29-7.36(m, 2H), 7.41 (t, J=7.40 Hz, 2H), 7.55-7.64 (m, 2H), 7.77 (d, J=7.58 Hz,2H). LCMS (ESI+) m/z: [MNa]⁺ calcd for C₂₀H₂₀N₂O₅Na⁺: 391.1, found:391.1.

Benzyl 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oate(18-102)

To a mixture of(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate(18-101) (80.0 g, 173 mmol, 1.0 equiv) in 1,2-dimethoxyethane (1.5 L)was added benzyl 2-hydroxyacetate (57.7 g, 49.3 mL, 347 mmol, 2.0equiv), cooled to 0° C. acetic acid (5.22 g, 86.8 mmol, 4.97 mL, 0.5equiv) added. The mixture was stirred at 0° C. for 1 h, sodium hydroxide(10 N, 17.3 mL, 1.0 equiv) added dropwise and stirred at 0° C. for 1 h,water (1.2 L) added and stirred at 0° C. for 2 h. The white precipitatesformed was filtered, washed with cold (5° C.) 1,2-dimethoxyethane:water(1:2, 500 mL) and methyl tert-butyl ester (500 mL), and dried undervacuum at 40° C. to give benzyl1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oate(18-102) (60.0 g, 69% yield). ¹H NMR (400 MHz, CDCl₃) δ ppm 3.82 (d,J=5.26 Hz, 2H), 4.16-4.29 (m, 3H), 4.45 (d, J=6.80 Hz, 2H), 4.83 (d,J=7.02 Hz, 2H), 5.16 (s, 2H), 5.45 (t, J=5.59 Hz, 1H), 7.08 (s, 1H),7.28-7.48 (m, 9H), 7.59 (d, J=7.23 Hz, 2H), 7.77 (d, J=7.45 Hz, 2H).LCMS (ESI+) m/z: [MNa]⁺ calcd for C₂₇H₂₆N₂O₆Na⁺: 497.2, found: 497.1.

Benzyl 2-((2-aminoacetamido)methoxy)acetate·HOBT salt (18-103)

To a mixture of benzyl1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oate(18-102) (60.0 g, 126 mmol, 1.0 equiv) in acetonitrile (1.08 L) wasadded DBU (9.53 mL, 63.2 mmol, 0.5 equiv) at 0° C. The mixture wasstirred at 25° C. for 4 h, cooled to 0° C., HOBt (34.1 g, 252 mmol, 2.0equiv) added and stirred at 0° C. for 1.5 h. It was warmed up to 25° C.,filtered, the filter cake washed with cold (5° C.) acetonitrile (200mL), and dried under vacuum to give benzyl2-((2-aminoacetamido)methoxy)acetate·HOBt (18-103) (46.0 g, 84% yield).¹H NMR (400 MHz, METHANOL-d4) δ ppm 3.68 (s, 2H), 4.21 (s, 2H), 4.79 (s,2H), 5.18 (s, 2H), 7.22-7.42 (m, 7H), 7.62-7.74 (m, 2H). LCMS (ESI−)m/z: [MH]⁻ calcd for C18H₃₃N₅O—: 269.1, found: 269.0.

(S)-Benzyl11-benzyl-3,6,9,12,15-pentaoxo-1-phenyl-2,18-dioxa-4,7,10,13,16-pentaazaicosan-20-oate(18-105)

To a mixture of benzyl 2-((2-aminoacetamido)methoxy)acetate·HOBt(18-103) (46.0 g, 118 mmol, 1.0 equiv) in acetonitrile (500 mL) wereadded(S)-11-benzyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-triazadodecan-12-oicacid (18-104) (45.0 g, 108 mmol, 0.9 equiv) andN¹-((ethylimino)methylene)-N³,N³-dimethylpropane-1,3-diaminehydrochloride (25.5 g, 133 mmol, 1.1 equiv) at 0° C. The mixture wasstirred at 0° C. for 3.5 h, water (200 mL) added and stirred at 0° C.for 2 h. The precipitates formed were filtered, washed with cold (5° C.)acetonitrile:water (1:2, 201 mL) and dried under vacuum to give(S)-benzyl11-benzyl-3,6,9,12,15-pentaoxo-1-phenyl-2,18-dioxa-4,7,10,13,16-pentaazaicosan-20-oate(18-105) (30.0 g, 34% yield). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 2.79 (dd,J=13.67, 9.92 Hz, 1H), 3.06 (dd, J=13.67, 4.41 Hz, 1H), 3.56-3.66 (m,3H), 3.69-3.81 (m, 3H), 4.15 (s, 2H), 4.51 (td, J=8.76, 4.52 Hz, 1H),4.63 (d, J=6.62 Hz, 2H), 5.03 (s, 2H) 5.13-5.17 (m, 2H), 7.03-7.46 (m,15H), 7.49-7.55 (m, 1H), 8.00-8.10 (m, 1H), 8.18 (d, J=7.94 Hz, 1H),8.36 (t, J=5.62 Hz, 1H), 8.62 (t, J=6.73 Hz, 1H).

(S)-16-Amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecan-1-oicacid (18-106)

To a mixture of (S)-benzyl11-benzyl-3,6,9,12,15-pentaoxo-1-phenyl-2,18-dioxa-4,7,10,13,16-pentaazaicosan-20-oate(18-105) (10.0 g, 15.4 mmol, 1.0 equiv) in tetrahydrofuran (210 mL) andH₂O (140 mL) was added Pd/C (10 wt %) (4.40 g, 1.2 equiv) at 25° C.under argon, the suspension was vacuumed and filled with H₂ three times,and stirred under H₂ (15 psi) at 25° C. for 2.5 h. Upon replacing H₂atmosphere with argon, it was filtered through a pad of Celite, and thefilter cake washed with water (80 mL) and ethanol (150 mL). The filtratewas concentrated to about 30 mL, ethanol (120 mL) was added, and themixture stirred at 0° C. for 2 h. The precipitates formed was collectedby filtration, washed with ethanol (30 mL), and dried under vacuum togive(S)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecan-1-oicacid (18-106) (4.00 g, 58% yield). ¹H NMR (400 MHz, D₂O) δ ppm 2.88-3.26(m, 2H), 3.64-4.16 (m, 8H), 4.55-4.71 (m, 3H), 7.11-7.50 (m, 5H). LCMS(ESI−) m/z: [MH]⁻ calcd for C18H₂₄N₅₀₇ ⁻: 422.2, found: 422.1.

(E)-4-((4-(2-Carboxyethyl)phenyl)amino)-4-oxobut-2-enoic acid (18-108)

To a mixture of furan-2,5-dione (4.16 g, 42.3 mmol, 1.0 equiv) in ether(42 mL) was added a solution of 3-(4-aminophenyl)propanoic acid (18-107)(7.00 g, 42.3 mmol, 1.0 equiv) and 2,6-dimethylpyridine (4.58 g, 4.98mL, 42.3 mmol, 1.0 equiv) in tetrahydrofuran (70 mL) at 25° C. Thereaction mixture was stirred at 65° C. for 0.5 h, cooled to 25° C.,filtered, the filter cake washed with methyl tert-butyl ether (150 mL),and dried under vacuum to give(E)-4-((4-(2-carboxyethyl)phenyl)amino)-4-oxobut-2-enoic acid (18-108)(11.0 g, 98% yield). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 12.12-13.09 (m,2H), 10.37 (s, 1H), 7.52 (d, J=8.4 Hz, 2H), 7.18 (d, J=8.4 Hz, 2H), 6.46(d, J=12.0 Hz, 1H), 6.30 (d, J=12.0 Hz, 1H), 2.78 (t, J=7.6 Hz, 2H),2.52 (s, 2H). LCMS (ESI−) m/z: [M−H]⁻ calcd for C₁₃H₁₂NO₅ ⁻: 262.0,found: 261.9.

3-(4-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanoic acid(18-109)

To a mixture of (E)-4-((4-(2-carboxyethyl)phenyl)amino)-4-oxobut-2-enoicacid (18-108) (11.0 g, 41.7 mmol, 1.0 equiv) in acetic anhydride (100mL) was added potassium; acetate (2.26 g, 22.9 mmol, 0.55 equiv) undernitrogen. The suspension was vacuumed and filled with nitrogen threetimes, stirred at 145° C. for 0.5 h, cooled to 20° C. and concentratedunder reduced pressure. The residue was diluted with ethyl acetate (300mL), washed with brine, dried over Na₂SO₄, filtered, concentrated, andthe residue purified by silica gel flash column chromatography elutingwith 33%-100% of ethyl acetate in petroleum ether containing 20%tetrahydrofuran to give3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanoic acid(18-109) (3.60 g, 29% yield). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 12.16 (s,1H), 7.33 (d, J=8.4 Hz, 2H), 7.20-7.24 (m, 2H), 7.16 (s, 2H), 2.86 (t,J=7.6 Hz, 2H), 2.57 (t, J=7.6 Hz, 2H). LCMS (ESI−) m/z: [M−H]-calcd forC₁₃H₁₀NO₄ ⁻: 244.0, found: 243.9.

2,5-Dioxopyrrolidin-1-yl3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanoate (18-110)

To a mixture of3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanoic acid(18-109) (3.60 g, 14.6 mmol, 1.0 equiv) in acetonitrile (72 mL) wereadded 1-hydroxypyrrolidine-2,5-dione (1.77 g, 15.4 mmol, 1.05 equiv) andN,N′-methanediylidenedicyclohexanamine (3.18 g, 15.4 mmol, 1.05 equiv).The reaction mixture was stirred at 25° C. for 3 h, filtered, thefiltrate containing 2,5-dioxopyrrolidin-1-yl3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl) propanoate (18-110)was used directly in the next step without work up and purification.LCMS (ESI+) m/z: [MH]⁺ calcd for C₁₇H₁₅N₂O₆ ⁺: 343.1, found: 343.1.

(S)-10-Benzyl-20-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazaicosan-1-oicacid (18-111)

To a mixture of 2,5-dioxopyrrolidin-1-yl3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanoate (18-110)(1.62 g, 4.72 mmol, 1.0 equiv) in acetonitrile (80 mL) was added asolution of(S)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecan-1-oicacid (18-106) (2.00 g, 4.72 mmol, 1.0 equiv) andN-ethyl-N-isopropylpropan-2-amine (0.658 mL, 3.78 mmol, 0.8 equiv) inH₂O (20 mL) at 25° C. The reaction mixture was stirred at 25° C. for 12h, filtered, concentrated and the residue purified by prep-HPLC to give(S)-10-benzyl-20-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazaicosan-1-oicacid (18-111) (0.78 g, 24% yield). ¹H NMR (400 MHz, DMSO-d₆) δ ppm8.53-8.64 (m, 1H), 8.31 (t, J=5.6 Hz, 1H), 8.23 (t, J=5.6 Hz, 1H), 8.16(d, J=8.0 Hz, 1H), 8.08 (t, J=5.2 Hz, 1H), 7.14-7.38 (m, 11H), 4.61 (d,J=6.8 Hz, 2H), 4.44-4.55 (m, 1H), 3.97 (s, 2H), 3.64-3.80 (m, 5H), 3.63(d, J=5.6 Hz, 1H), 3.06 (dd, J=13.6, 4.4 Hz, 1H), 2.78-2.89 (m, 3H),2.53-2.60 (m, 1H). LCMS (ESI−) m/z: [MH]-calcd for C₃₁H₃₃N₆O₁₀ ⁻: 649.2,found: 649.3.

Prep-HPLC Condition:

-   -   Instrument: Gilson 281 semi-preparative HPLC system    -   Mobile phase: A: H₂O; B: ACN    -   Column: Agela DuraShell C18 250*70 mm*10 um    -   Flow rate: 130 mL/min    -   Monitor wavelength: 220&254 nm

Time B % 0.0 10 20.0 40 20.1 40 20.2 100 26.2 100 26.3 10 27.5 10

(S)-2-(2-(2-(3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanamido)acetamido)acetamido)-N-(2-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide(18-112)

A mixture of(S)-10-benzyl-20-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazaicosan-1-oicacid (18-111) (246 mg, 0.378 mmol, 1.0 equiv),(1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione hydrochloride (8-71) (170 mg, 0.378 mmol, 1.0 equiv),4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholin-4-iumtetrafluoroborate (124 mg, 0.378 mmol, 1.0 equiv) and 4-methylmorpholine(0.166 mL, 1.51 mmol, 4.0 equiv) in N, N-dimethylformamide (5 mL) wasstirred at 25° C. for 1 h. The mixture was filtered and the filtratepurified by prep-HPLC to give(S)-2-(2-(2-(3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanamido)acetamido)acetamido)-N-(2-(((2-(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide(18-112) (130 mg, 30% yield). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.39 (s,1H), 8.32-8.37 (m, 1H), 8.12-8.22 (m, 2H), 8.04 (t, J=5.6 Hz, 1H), 7.78(d, J=10.8 Hz, 1H), 7.26-7.34 (m, 3H), 7.18-7.26 (m, 6H), 7.16 (s, 2H),6.51 (s, 1H), 5.28-5.50 (m, 3H), 4.92 (d, J=18.8 Hz, 1H), 4.67 (d, J=6.8Hz, 2H), 4.51 (d, J=4.0 Hz, 1H), 3.90-4.05 (m, 2H), 3.64-3.83 (m, 5H),3.62 (d, J=5.6 Hz, 1H), 3.21-3.27 (m, 1H), 3.00-3.13 (m, 2H), 2.89-2.99(m, 1H), 2.76-2.87 (m, 3H), 2.46 (s, 3H), 2.38 (s, 3H), 1.75-2.01 (m,3H), 1.58 (s, 3H), 0.87 (t, J=7.2 Hz, 3H). ¹⁹F NMR (376 MHz, DMSO-d₆) δppm −111.87. LCMS (ESI+) m/z: [MH]⁺ calcd for C₅₆H₅₇FN₉O₁₃: 1082.4,found: 1082.3.

Prep-HPLC Condition:

-   -   Instrument: Gilson 281 semi-preparative HPLC system    -   Mobile phase: A: H₂O; B: ACN    -   Column: Phenomenex C18 75*30 mm*3 um    -   Flow rate: 25 mL/min    -   Monitor wavelength: 220&254 nm

Time B % 0.0 20 8.0 50 8.1 50 8.2 100 10.2 100 10.3 20 11.5 20

Example 19N—((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzamide(19-113)

Example 19 (19-113) was made in a similar fashion to Example 18(18-112). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.87 (t, J=5.6 Hz, 1H), 8.69(t, J=6.4 Hz, 1H), 8.32-8.41 (m, 2H), 8.10-8.19 (m, 2H), 7.90 (d, J=7.6Hz, 1H), 7.83-7.86 (m, 1H), 7.78 (d, J=10.8 Hz, 1H), 7.55-7.61 (m, 1H),7.48-7.53 (m, 1H), 7.31 (s, 1H), 7.18-7.26 (m, 6H), 7.13-7.18 (m, 1H),6.51 (s, 1H), 5.30-5.47 (m, 3H), 4.92 (d, J=18.8 Hz, 1H), 4.67 (d, J=7.2Hz, 2H), 4.51 (dd, J=8.0, 4.4 Hz, 1H), 3.84-4.04 (m, 4H), 3.71-3.83 (m,3H), 3.54-3.68 (m, 1H), 3.25 (s, 1H), 2.88-3.15 (m, 3H), 2.82 (dd,J=13.6, 9.6 Hz, 1H), 2.38 (s, 3H), 1.73-2.04 (m, 3H), 1.58 (s, 3H), 0.87(t, J=7.2 Hz, 3H). ¹⁹F NMR (376 MHz, DMSO-d₆) δ ppm −111.87. LCMS (ESI+)m/z: [MH]⁺ calcd for C₅₄H₅₃FN₉O₁₃ ⁺: 1054.3, found: 1054.3.

Example 20N—((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide(20-114)

Example 20 (20-114) was made in a similar fashion to Example 18(18-112). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.69 (t, J=6.4 Hz, 1H), 8.39(s, 1H), 8.34 (J=5.6 Hz, 1H), 8.14 (d, J=8.0 Hz, 1H), 8.04 (t, J=5.6 Hz,1H), 7.99 (t, J=5.6 Hz, 1H), 7.78 (d, J=10.8 Hz, 1H), 7.31 (s, 1H),7.20-7.28 (m, 4H), 7.14-7.20 (m, 1H), 6.98 (s, 2H), 6.51 (s, 1H),5.35-5.48 (m, 3H), 4.92 (d, J=18.8 Hz, 1H), 4.67 (d, J=6.8 Hz, 2H),4.46-4.54 (m, 1H), 3.90-4.04 (m, 2H), 3.70-3.85 (m, 3H), 3.55-3.67 (m,3H), 3.33-3.38 (m, 2H), 3.26 (d, J=2.0 Hz, 1H), 2.87-3.11 (m, 3H), 2.81(dd, J=13.6, 9.6 Hz, 1H), 2.39 (s, 3H), 2.08 (t, J=7.6 Hz, 2H),1.76-2.01 (m, 3H), 1.58 (s, 3H), 1.40-1.51 (m, 4H), 1.12-1.23 (m, 2H),0.87 (t, J=7.2 Hz, 3H). ¹⁹F NMR (376 MHz, DMSO-d₆) δ ppm −111.88. LCMS(ESI+) m/z: [MH]⁺ calcd for C₅₃H₅₉FN₉O₁₃ ⁺: 1048.4, found: 1048.5.

Example 21(S)-2-(2-(2-(3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanamido)acetamido)acetamido)-N-(2-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)oxy)ethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide(21-120) (FIG. 26)

(2S)-2-[[2-[[2-[3-[4-(2,5-Dioxopyrrol-1yl)phenyl]propanoylaminolacetyl]amino]acetyl]amino]-3-phenyl-propanoicacid (21-116)

A mixture of (S)-2-(2-(2-aminoacetamido)acetamido)-3-phenylpropanoicacid (21-115) (5.03 g, 14.6 mmol, 1.0 equiv) andN-ethyl-N,N-diisopropylamine (1.52 g, 11.7 mmol, 2.05 mL, 0.8 equiv) inH₂O (25 mL) was added dropwise to a mixture of 2,5-dioxopyrrolidin-1-yl3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanoate (18-110)(4.10 g, 14.6 mmol, 1.0 equiv) in acetonitrile (75 mL). The reactionmixture was stirred at 25° C. for 5 h, filtered and the filtratepurified by prep-HPLC to give(2S)-2-[[2-[[2-[3-[4-(2,5-dioxopyrrol-1-yl)phenyl]propanoylamino]acetyl]amino]acetyl]amino]-3-phenyl-propanoicacid (21-116) (3.32 g, 40% yield). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.21(t, J=5.6 Hz, 1H), 8.07 (t, J=5.6 Hz, 1H), 7.87 (d, J=7.6 Hz, 1H), 7.32(d, J=8.4 Hz, 2H), 7.06-7.27 (m, 9H), 4.29 (d, J=5.2 Hz, 1H), 3.61-3.77(m, 4H), 3.19 (s, 1H), 3.03 (d, J=4.8 Hz, 1H), 2.86 (t, J=8.0 Hz, 3H),2.67 (s, 1H). LCMS (ESI−) m/z: [MH]⁺ calcd for C₂₆H₂₅N₄O₇ ⁻: 505.1,found: 505.2.

Prep-HPLC Condition:

-   -   Instrument: Gilson 281 semi-preparative HPLC system    -   Mobile phase: A: H2O; B: ACN    -   Column: Phenomenex C18 250*100 mm 10 u    -   Flow rate: 260 mL/min    -   Monitor wavelength: 220&254 nm

Time B % 0.0 1 20.0 40 20.1 40 20.2 100 25.2 100 25.3 1 26.5 1

(9H-Fluoren-9-yl)methyl(2-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)oxy)ethoxy)methyl)amino)-2-oxoethyl)carbamate(21-118)

To a mixture of(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethoxy)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (5-48) (1.25 g, 2.60 mmol, 1.0 equiv) in dichloromethane(37 mL) at 25° C. was added Scandium(III) trifluoromethanesulfonate(50.5 mg, 0.260 mmol, 0.1 equiv), and[[2-(9H-fluoren-9-ylmethoxycarbonylamino)acetyl]amino]methyl acetate(21-117) (1.44 g, 3.90 mmol, 1.5 equiv) in three portions over 1.5 h.The reaction mixture was stirred at 30° C. for 36 h, quenched with water(100 mL) and extracted with dichloromethane (3×100 mL). The combinedorganic phases were washed with brine, dried over Na₂SO₄, filtered,concentrated, and the residue purified by silica gel flash columnchromatography eluting with 10%-100% of dichloromethane in ethyl acetateto give (9H-fluoren-9-yl)methyl(2-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)oxy)ethoxy)methyl)amino)-2-oxoethyl)carbamate (21-118) (1.00 g, 45%yield). ¹H NMR (400 MHz, DMSO-D₆) δ ppm 8.90 (br t, J=6.60 Hz, 1H),7.80-7.91 (m, 2H), 7.73 (d, J=11.13 Hz, 1H), 7.56-7.63 (m, 2H),7.23-7.44 (m, 6H), 6.53 (s, 1H), 5.32-5.53 (m, 3H), 4.96 (br dd, J=9.60,4.10 Hz, 1H), 4.62-4.83 (m, 2H), 4.44-4.59 (m, 1H), 4.20-4.32 (m, 1H),4.05-4.19 (m, 3H), 3.91-4.00 (m, 1H), 3.57-3.78 (m, 5H), 3.14-3.28 (m,1H), 2.86-3.08 (m, 1H), 2.34 (s, 3H), 1.85 (br dd, J=7.03, 4.46 Hz, 3H),0.78-0.94 (m, 3H). ¹⁹F NMR (376 MHz, DMSO-d₆) δ ppm −111.56. LCMS (ESI+)m/z: [MH]⁺ calcd for C₄₄H₄₂FN₄O₉ ⁺: 789.3, Found: 789.3.

2-Amino-N-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)oxy)ethoxy)methyl)acetamide (21-119)

To a mixture of (9H-fluoren-9-yl)methyl(2-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)oxy)ethoxy)methyl)amino)-2-oxoethyl)carbamate(21-118) (1.00 g, 1.27 mmol, 1.0 equiv) in N,N-dimethylformamide (20 mL)was added piperidine (0.125 mL, 1.27 mmol, 1.0 equiv). The reactionmixture was stirred at 0° C. for 1 h, filtered and the filtrate purifiedby prep-HPLC to give2-amino-N-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)oxy)ethoxy)methyl)acetamide(21-119) (360 mg, 45% yield). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.78 (t,J=6.8 Hz, 1H), 7.66-7.81 (m, 1H), 7.32 (s, 1H), 6.52 (s, 1H), 5.35-5.46(m, 3H), 5.01 (dd, J=9.2, 3.6 Hz, 1H), 4.59-4.83 (m, 2H), 3.92-4.03 (m,1H), 3.77 (m, 1H), 3.59-3.71 (m, 2H), 3.20-3.29 (m, 2H), 3.16 (s, 2H),2.92-3.05 (m, 1H), 2.59-2.84 (m, 1H), 2.29-2.41 (m, 3H), 1.72-2.09 (m,3H), 0.78-0.97 (m, 3H). ¹⁹F NMR (376 MHz, DMSO-d₆) δ ppm −111.54. LCMS(ESI+) m/z: [MH]⁺ calcd for C₂₉H₃₂FN₄O₇ ⁺: 567.2, found: 567.3.

Prep-HPLC Conditions

-   -   Instrument: Gilson 281 semi-preparative HPLC system    -   Mobile phase: A: 10 mM NH₄HCO₃ in H₂O; B: ACN    -   Column: Phenomenex C18 75*30 mm*3 um    -   Flow rate: 25 mL/min    -   Monitor wavelength: 220&254 nm

Time B % 0.0 10 8.0 40 8.1 40 8.2 100 10.2 100 10.3 10 11.5 10

(S)-2-(2-(2-(3-(4-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanamido)acetamido)acetamido)-N-(2-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)oxy)ethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide (21-120)

A mixture of(2S)-2-[[2-[[2-[3-[4-(2,5-dioxopyrrol-1-yl)phenyl]propanoylamino]acetyl]amino]acetyl]amino]-3-phenyl-propanoicacid (21-116) (160 mg, 0.317 mmol, 1.0 equiv),2-amino-N-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)oxy)ethoxy)methyl)acetamide(21-119) (180 mg, 317 umol, 1.0 equiv),4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholin-4-iumtetrafluoroborate (104 mg, 0.317 mmol, 1.0 equiv) and 4-methylmorpholine(0.139 mL, 1.27 mmol, 4.0 equiv) in N,N-dimethylformamide (3.6 mL) wasstirred at 25° C. for 1 h, filtered and the filtrate purified byprep-HPLC to give(S)-2-(2-(2-(3-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)propanamido)acetamido)acetamido)-N-(2-(((2-(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]py-rano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)oxy)ethoxy)methyl)amino)-2-oxoethyl)-3-phenyl-propanamide(21-120) (80.0 mg, 23% yield). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.69 (t,J=6.8 Hz, 1H), 8.28 (t, J=5.6 Hz, 1H), 8.19 (t, J=5.6 Hz, 1H), 8.10 (d,J=8.0 Hz, 1H), 8.03 (t, J=5.6 Hz, 1H), 7.76 (d, J=11.2 Hz, 1H),7.26-7.35 (m, 3H), 7.13-7.25 (m, 8H), 6.52 (s, 1H), 5.34-5.53 (m, 4H),5.03 (dd, J=8.8, 4.00 Hz, 1H), 4.63-4.81 (m, 2H), 4.41-4.57 (m, 1H),3.90-4.03 (m, 1H), 3.74-3.84 (m, 3H), 3.54-3.74 (m, 6H), 3.16-3.30 (m,2H), 2.93-3.07 (m, 2H), 2.73-2.88 (m, 3H), 2.42-2.48 (m, 3H), 2.37 (s,3H), 1.80-2.03 (m, 3H), 0.87 (t, J=7.2 Hz, 3H). ¹⁹F NMR (376 MHz,DMSO-d₆) δ ppm −111.53. LCMS (ESI+) m/z: [MH]⁺ calcd for C₅₅H₅₆FN₈O₁₃ ⁺:1055.4, found: 1055.3.

Prep-HPLC Conditions

-   -   Instrument: Gilson 281 semi-preparative HPLC system    -   Mobile phase: A: H₂O; B: ACN    -   Column: Phenomenex C18 75*30 mm*3 um    -   Flow rate: 60 mL/min    -   Monitor wavelength: 220&254 nm

Time B % 0.0 25 8.0 55 8.1 55 8.2 100 10.2 100 10.3 25 11.5 25

Example 22N—((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)oxy)-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzamide(22-121)

Example 22 (22-121) was made in a similar fashion to Example 21(21-120). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.86 (t, J=5.60 Hz, 1H), 8.67(br t, J=6.56 Hz, 1H), 8.29 (br t, J=5.66 Hz, 1H), 8.05-8.17 (m, 2H),7.89 (d, J=7.99 Hz, 1H), 7.83 (s, 1H), 7.75 (d, J=10.97 Hz, 1H),7.54-7.61 (m, 1H), 7.47-7.53 (m, 1H), 7.33 (s, 1H), 7.11-7.25 (m, 7H),6.52 (s, 1H), 5.34-5.48 (m, 4H), 5.03 (br dd, J=8.82, 3.58 Hz, 1H),4.63-4.79 (m, 2H), 4.44-4.54 (m, 1H), 3.96 (br dd, J=7.03, 4.17 Hz, 1H),3.88 (br d, J=5.72 Hz, 2H), 3.57-3.81 (m, 7H), 3.19-3.27 (m, 1H),2.93-3.08 (m, 2H), 2.78 (br dd, J=13.65, 9.83 Hz, 1H), 2.45 (br s, 1H),2.36 (s, 3H), 1.81-2.02 (m, 3H), 0.83-0.91 (m, 3H). ¹⁹F NMR (376 MHz,DMSO-d₆) δ ppm −111.54. LCMS (ESI+) m/z: [MH]⁺ calcd for C₅₃H₅₂FN₈O₁₃ ⁺:1027.3, found: 1027.3.

Example 23N—((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)oxy)-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide(23-122)

Example 23 (23-122) was made in a similar fashion to Example 21(21-120). ¹H NMR (400 MHz, DMSO-D₆) δ ppm 8.63-8.74 (m, 1H), 8.28 (t,J=5.63 Hz, 1H), 8.09 (d, J=8.13 Hz, 1H), 8.04 (t, J=5.63 Hz, 1H), 7.98(t, J=5.63 Hz, 1H), 7.77 (d, J=11.01 Hz, 1H), 7.34 (s, 1H), 7.11-7.26(m, 5H), 6.98 (s, 2H), 6.52 (s, 1H), 5.31-5.58 (m, 4H), 5.05 (br dd,J=9.13, 4.00 Hz, 1H), 4.61-4.82 (m, 2H), 4.40-4.55 (m, 1H), 3.90-4.04(m, 1H), 3.73-3.84 (m, 3H), 3.60-3.73 (m, 5H), 3.53-3.60 (m, 1H),3.33-3.37 (m, 2H), 3.21-3.27 (m, 1H), 2.94-3.08 (m, 2H), 2.77 (dd,J=13.70, 9.82 Hz, 1H), 2.46-2.48 (m, 1H), 2.38 (s, 3H), 2.05-2.12 (m,2H), 1.82-2.01 (m, 3H), 1.45 (dq, J=14.13, 7.00 Hz, 4H), 1.10-1.22 (m,2H), 0.87 (t, J=7.32 Hz, 3H). ¹⁹F NMR (376 MHz, DMSO-d₆) δ ppm −111.54.LCMS (ESI+) m/z: [MH]⁺ calcd for C₅₂H₅₅FN₈O₁₃ ⁺: 1021.4, found: 1021.4.

Example 24 CTG Assays (Jeko-1 and MDA-MB-468)

The CTG assay is a method of determining the number of viable cells inculture based on quantitation of the ATP present, an indicator ofmetabolically active cells. The cell assay requires the addition of asingle reagent, Cell Titer Glo, in which cells are lysed and generationof a luminescent signal is produced. The luminescent signal isproportional to the amount of ATP present. The amount of ATP is directlyproportional to the number of cells present in culture. For our assay,cells were ensured to be in log-phase for either Jeko-1 or MDA-MB-468.Cells were transferred to 96 wells and treated with compounds inthree-fold serial dilution starting from 1 μM to 0.0000508 μM (10 pointsdilution), for 72 h. Cell viability was analyzed with CellTiter-Glo®Luminescent Cell Viability Assay (Promega) following manufactures'instruction. Percentage of viable cells in each compound concentrationwas determined by normalizing with the luminescence of vehicle controland plotted into percentage of viability versus dose response curve bynonlinear fit in GraphPad Prism software. Compound ICso was calculatedas the concentration of compound killing 50% of cells. Representativeassay results are summarized in Table 1.

Example 25 Human Hepatocyte Clearance (HHEP CL)

Suspensions of human hepatocytes (from 10 mixed gender human donors,final concentration 0.5×10⁶ cell/mL) in Williams' E medium wereincubated for 90 min with a test compound (0.90% acetonitrile and 0.10%DMSO, final concentration 1 mM) and positive controls (7-Ethoxycoumarin,7-Hydroxycoumarin, 0.90% acetonitrile and 0.10% DMSO, finalconcentration 3 mM), with constant shaking at about 600 rpm at 37° C. inan incubator at 5% CO₂ and 95% humidity. The total volume of incubationwas 200 μl. A sample (25 mL) was taken out at TO, 15, 30, 60 and 90 min,which was added intermediately to the ice-cold stop solution(acetonitrile with 200 ng/mL of tobutamide and labetalol as internalstandard) (125 μl), and vortexed at 500 rpm for 10 min, centrifuged at3220×g for 20 min at 4° C. Analytical plates are sealed and stored at 4°C. until LCMS analysis. Viability of hepatocytes at pre-incubation wasdetermined to be 84.5%. Representative assay results are summarized inTable 1.

Example 26 Human Liver Microsome Clearance (HLM CL)

Working solution was prepared by adding 5 μL of compound and controlstock solution (10 mM in dimethyl sulfoxide, DMSO) to 495 μL ofacetonitrile (ACN) (intermediate solution concentration: 100 μM, 99% CANand 1% DMSO. The appropriate concentrations of microsome workingsolutions were prepared in 100 mM potassium phosphate buffer. Afterreaction plates containing mixtures of compound and microsomes werepre-incubated at 37° C. for 10 min, 98 mL of 2 mM of NADPH and 2 mM ofMgCl₂ solution was added to start the reaction. The final concentrationsof incubation medium were as follows: microsome—0.5 mg protein/mL, testcompound/control compound—1 mM, NADPH—1 mM, MgCl₂ —1 mM, acetonitrile0.99%, DMSO 0.01%. Incubations were performed at 37° C. for 60 min.Samples were taken out at TO, T5, T15, T30, T45 and T60, which was addedintermediately to the ice-cold stop solution (acetonitrile with 200ng/mL of tobutamide and labetalol as internal standard) (125 μl), shakenfor 10 min, centrifuged at 4000 rpm for 20 min at 4° C. Analyticalplates were analyzed by LCMS. Representative assay results aresummarized in Table 1.

Human liver microsome clearance assay assess metabolism by thecytochrome P450 system (phase I enzymes). These enzymes oxidizesubstrates by incorporating oxygen atoms into hydrocarbons, thus causingthe introduction of hydroxyl groups, or N- O- and S-dealkylation ofsubstrates and forming more polar products easier to be cleared. Humanhepatocyte clearance assay measures more broadly the overall cellularmetabolism of the test compound (phase I and phase II enzyme pathways).Phase II enzymes catalyze the conjugation reaction of xenobioticmetabolites and charged species, such as glutathione, sulfate, glycine,or glucuronic acid to form even more polar compounds for easierclearance.

The payloads with higher intrinsic clearance may provide bettertherapeutic index due to their potential lower systemic plasma exposure.(Maderna, A.; Doroski, M; Subramanyam, C.; Porte, A.; Leverett, C. A.;Vetelino, B. C.; Chen, Z.; Risley, H.; Parris, K.; Pandit, J.; Varghese,A. H.; Shanker, S.; Song, C.; Sukuru, S. C. K.; Farley, K. A.; Wagenaar,M. M.; Shapiro, M. J.; Musto, S.; Lam, M−H.; Loganzo, F.; O'Donnell, C.J. “Discovery of cytotoxic dolastatin 10 analogues with N-terminalmodifications” Journal Medicinal Chemistry, 2014, 57, 10527-10543). InTable 1, the payloads with higher intrinsic clearance likely have animproved safety profile because the payload, which is potentially toxicto healthy cells, is quickly removed from the plasma, decreasing itschance of interacting with healthy cells.

Example 27 PAMPA (Parallel Artificial Membrane Permeability Assay)

PAMPA is a method which determines the permeability of substances from adonor compartment, through a lipid-infused artificial membrane into anacceptor compartment. See Ottaviani, G.; Martel, S.; Carrupt, P-A.“Parallel Artificial Membrane Permeability Assay: A New Membrane for theFast Prediction of Passive Human Skin Permeability”, Journal ofMedicinal Chemistry, 2006, 49 (13), 3948-3954). A multi-well microtitreplate is used for the donor and a membrane/acceptor compartment isplaced on top; the whole assembly is commonly referred to as a“sandwich”. At the beginning of the test, the drug is added to the donorcompartment, and the acceptor compartment is drug-free. After anincubation period which may include stirring, the sandwich is separated,and the amount of drug is measured in each compartment. Mass balanceallows calculation of drug that remains in the membrane.

The PAMPA was performed by Pion Inc using the GIT-0 lipid and 5 μM donorsolution in pH 5.0 and pH 7.4 PRISMA buffer (containing 0.05% DMSO). Thehigher PAMPA data has been associated with better bystander killing.(Ogitani Y.; Hagihara K.; Oitate, M.; Naito, H.; Agatsuma T. “Bystanderkilling effect of DS-8201a, a novel anti-human epidermal growth factorreceptor 2 antibody-drug conjugate, in tumors with human epidermalgrowth factor receptor 2 heterogeneity” Cancer Science, 2016, 107 (7),1039-1046).

Representative assay results are summarized in Table 1. Higherpermeability is important because it implies greater potential for“bystander killing”. That is, once the payload has neutralized a tumorcell, a more permeable payload is more likely to escape the neutralizedtumor cell, then imbed in a neighboring tumor cell. Once there, it canneutralize the tumor cell, escape, embed in another neighboring tumorcell, and repeat the process.

TABLE 1 PAMPA Pe JeKo-1 MDA- (10⁻⁶ cm/s) HHEP Cl HLM Cl IC₅₀ MB-468Compound pH 5.0/7.4 (T_(1/2)) (T_(1/2)) (nM) IC₅₀ (nM) Dxd 21.9/5.0 48.7 (79.0)  8.6 (>145) 1.42 11.6 Exatecan 37.2/5.7  59.3 (65.0) 11.7(106) 0.59 6.2  3-34 78.3/8.2  79.2 (48.7) 32.1 (38.8) 0.59 6.0  3-3566.1/11.1  50.4 (76.5) 20.2 (61.8) 0.46 4.7  5-47 76/.1/11.6  53.7(71.7) 17.3 (72.2) 1.5 6.3  5-48 69.0/9.0  75.5 (51.1) 17.4 (71.7) 0.688.3  7-59 ND  69.4 (55.5) 33.7 (37.0) 2.9 14.8  7-60 ND  64.5 (59.7)40.8 (30.6) 2.1 14.9  8-71 67.7/8.6  81.7 (47) 41.9 (30) 0.44 6.1  8-7280.8/8.4  79.1 (49) 35.9 (35) 1.5 7.7  9-74 36.1/4.4  57.5 (67.0) 22.7(54.9) 4.1 57.7 10-75 46.8/3.9  40.8 (94.4) 27.2 (45.9) 58.1 467 13-8464.4/7.6 111.9 (34) 58.9 (21) 0.61 5.9 13-85 69.7/6.0  75.7 (51) 58.3(21) 2.6 36.2 14-91 67.8/5.6  63.4 (60.8) 46.5 (26.8) 1.4 17.6 14-9269.1/4.5  51.3 (75.1) 19.2 (65.0) 1.2 14.2 16-95 63.1/5.5  29.6 (130)25.4 (49.1) 1.3 13.7 17-96 62.6/4.3  47.1 (82) 71.3 (17.5) 2.1 22 Dxd:deruxtecan; HHEP Cl: human hepatocytes intrinsic clearance, mL/min/Kg;HHEP t_(1/2), min. HLM: human liver microsome clearance, mL/min/Kg. ND:Not determined.

Example 28

Novel and diversified anti-ROR-1 specific monoclonal antibodies weredeveloped to bind to multiple regions of the ROR-1 extracellular domain(ECD) by employing an antibody development campaign using threestrategies: (1) mice of cohort 1 were immunized using full length ROR-1ECD; (2) mice of cohorts 2 and 3 were immunized with the ROR-1 IgG-likedomain; and (3) mice of cohort 4 were immunized with a short region ofthe human IgG-like sequence of ROR-1. After immunization of the mice,monoclonal antibodies were generated using conventional approaches.Briefly, unique variable heavy and light chain pairs from hybridoma andphage display campaigns were cloned into vectors designed to expressfull length antibodies as IgGs in HEK293 cells under the control of aCMVpromoter. Antibody expression vectors were complexed withpolyethylenimine and transfected into HEK293 cultures. After 5 days ofshaking at 37° C. in 293 cell culture media, antibodies were captured onagarose-based protein A resin. After several stringent washes,antibodies were eluted in glycine solution, pH 3, neutralized withHepes, pH 9, and buffer exchanged into PBS.

Several monoclonal antibodies were developed using these approaches andthe generated antibodies were subjected to additional screening toassess specific characteristics of the antibodies. To fully evaluate thecharacteristics of the novel antibodies several assays were performed.First, confirmation of antibody binding to the ROR-1 epitope wasconfirmed both biochemically, as well as, in ROR-1 positive cell lines.The specificity of binding was assessed biochemically by screeningbinding to human ROR-2 protein, rodent ROR-1 protein, as well as, incell-based assays. Further screening parameters included analyses ofantibody internalization, epitope binning against known anti-ROR-1antibodies (UC961 and 4a5), binding to human ROR-1 Ig-like domain,thermal shift, and assessment for self-interaction with Affinity-CaptureSelf-Interaction Nanoparticle Spectroscopy (AC-SINS).

Example 29

A cell binding saturation assay was developed to evaluate how well theanti_ROR-1 antibodies developed in Example 28 bound to endogenouslyexpressed extracellular ROR-1 protein on cell lines. More specifically,the anti-ROR-1 monoclonal antibodies developed in Example E, e.g.,ATX-P-875, ATX-P-885, and ATX-P-890, were analyzed in a cellular bindingassay. Briefly, two ROR-1 positive cell lines, JeKo-1 and MDA-MB-468,were incubated in a titration series concentration of each antibodyconstruct. Cells were then washed and subjected to secondary antibodystaining and detection by flow cytometry. Mean fluorescence (MFI) wasdetermined by analysis on cytometer software. The binding of ATX-P-875,ATX-P-885, and ATX-P-890 was compared to cell binding saturation datafor the monoclonal anti-ROR-1 antibody UC961. (See FIG. 27 ). As shownin FIG. 27 , the cell binding saturation for antibodies ATX-P-875,ATX-P-885, and ATX-P-890 were comparable to the cell binding saturationfor UC961 though a greater concentration of ATX-P-875 was needed toachieve saturation, as compared to UC961. ATX-P-890 and ATX-P-885 wereas good or improved, respectively, compared to UC-961 in concentrationsneeded to achieve binding saturation. Comparable saturation to UC961demonstrates that the anti-ROR-1 antibodies ATX-P-875, ATX-P-885, andATX-P-890 have a similar affinity to the human ROR-1 target as aclinically approved antibody UC-961.

Example 30

After a saturating concentration (74 nM) was determined in the bindingassay, the anti-ROR-1 antibodies developed herein (ATX-P-875, P-885,P-890) were evaluated for their capacity to internalize the ROR-1receptor on human ROR-1 positive cells (JeKo-1 and MDA-MB-468). Briefly,the ROR positive cell lines were incubated with antibody at supersaturating conditions so as to bind all available ROR-1 receptors.Excess antibody was washed off and the cells were incubated at 37° C.for a designated amount of time over a four-hour time course. At the endof each time point, internalization was stopped by placing an aliquot ofcells on ice. The antibody remaining on the surface was detected using alabeled secondary antibody and flow cytometry. Percent internalizationwas calculated based on time zero, and time zero was assumed that 100%of available receptors are on the cell surface. The results in FIG. 28demonstrate that all antibodies internalize ROR-1 receptor on JeKo-1 andMDA-MB-468 cells by a reduction of at least 75% over 4 hours.Unexpectedly, in MDA-MB-468, internalization of two of the anti-ROR-1antibodies (ATX-P-875 and ATX-P-890) was improved over the clinicallyused UC961 anti-ROR-1 antibody providing evidence that the ATX-P-875 andATX-P-890 antibodies have an improved ability to internalize the ROR-1receptor from the surface of solid tumors.

Example 31

Cellular binning was also employed to determine if monoclonal antibodiesATX-P-875, ATX-P-885, and ATX-P-890 bound to the same epitopes asconventionally known anti-ROR-1 binding monoclonal antibodies UC961 and4A5 (controls). In Step 1 of the cellular binning experiments,ATX-P-875, ATX-P-885, and ATX-P-890 monoclonal antibodies wereseparately incubated with ROR-1 expressing cells (MDA-MB-468) at variousamounts. In Step 2, a fluorescently labeled secondary antibodyrecognizing the novel antibodies was incubated with the samples. Andfinally in Step 3, the ROR-1 expressing cells coated with ATX-P-875,ATX-P-885, and ATX-P-890 were incubated with a saturating dose oflabeled UC961 (Dy650-UC 961) or 4A5 antibody (PE 4A5) and analyzed byflow cytometry. The UC961 and 4A5 staining signal was then compared tothe novel antibody staining signal to determine if the ATX-P-875,ATX-P-885, and ATX-P-890 antibodies bound the same epitope as the knownROR-1 binding antibodies UC961 and 4A5. FIG. 29A below shows thestaining profile expected if the ATX-P-875, ATX-P-885, and ATX-P-890antibodies bound the same epitope as the UC961 and 4A5 antibodies. FIG.29B, shows the expected profile if the ATX-P-875, ATX-P-885, andATX-P-890 antibodies bound to a separate epitope on ROR-1 than the UC961or 4A5 antibodies. Briefly, if binding the same epitope, increased novelantibody concentration would block the binding of prelabeled competitorantibody, thereby reducing the signal of the competitor at higherconcentrations. In the case antibodies bound separate epitopes, eachantibody, novel and competitor, would have increased staining withincreased dose as there would be no competition for binding to thereceptor. The cellular binning data obtained in MDA-MB-468 cellsindicated that ATX-P-885 appreciably bound the same epitope as UC961 andboth ATX-P-875 and ATX-P-890 appreciably bound the same epitope as 4A5.(See FIG. 29C, 29D and FIG. 30 ). The ability of the antibodiesdeveloped herein to bind distinct ROR-1 epitopes provides theopportunity to regulate the target in a variety of ways.

Example 32

Biochemical binning by SPR was also evaluated for the anti-ROR1antibodies (ATX-P-875, P-885, P-890) as compared against controlanti-ROR-1 antibodies UC961 and 4a5. In these experiments 10 ug/ml ofpurified clonal protein of Hu/Cy/Rh ROR1-His was covalently coupled tothe HC30M chip. Individual dilutions of each antibody at 10 μg/mL wereinjected over the chip and binding was evaluated by Carterra SPR.Unexpectedly the data demonstrate that there are 3 distinct bindingepitopes between ATX-P-875, ATX-P-885, and ATX-P-890 with ATX-885 beingthe only antibody to impart partial blocking to the UC961 antibody (SeeFIG. 31 ). Cellular binning only evaluated the antibodies ability toblock either UC961 or 4a5, two clinically used ROR-1 antibodies.Biochemical SPR evaluation also tested the antibodies' ability to blockeach other and found that ATX-P-875 was able to block the binding of 4a5as well as ATX-P-885, while still not being able to block UC961.

Example 33

Antibody characterization of ATX-P-875, ATX-P-885, and ATX-P-890, ascompared to UC961, are summarized in FIG. 30 and TABLES 2-7. An initialassessment of antibody developability was performed by AC-SINS toevaluate the potential for self-interaction (FIG. 30 ). Controlantibodies Adalimumab and Rituximab show expected low shift andInfliximab shows an expected high shift. The anti-ROR-1 antibodiesdeveloped herein, ATX-P-875, ATX-P-885, and ATX-P-890, are in line withcontrol antibodies that do not show significant self-interaction and arenot likely to pose a significant developability risk. Additionally, thebinding characteristics of monoclonal antibodies ATX-P-875, ATX-P-885,and ATX-P-890 were compared to the binding characteristics for UC961 inadditional experiments. TABLES 2-5 provide this antibodycharacterization data in comparison to the known ROR-1 binding antibodyUC961 including tabled results for biochemical binding to purifiedproteins and measured by SPR (TABLES 2-5), cellular binding to ROR-1positive cell lines JeKo-1 and MDA-MB-468 (EC50) (TABLE 6), and cellularinternalization (% internalized) (TABLE 7). Of particular note, it isbelieved that the reduced affinity of ATX-P-885 (KD: 1.09E-08) comparedto UC961 and other anti-ROR-1 antibodies (ATX-P-875 and ATX-P-890) canprovide an unexpected therapeutic benefit. It is contemplated that bybinding less tightly to the ROR-1 epitope, the ATX-P-885 antibody canpenetrate further into the tumor to reach more distant cells expressingROR-1 target.

TABLE 2 HUMAN/CYNO/RHESUS ROR-1 BINDING Hu/Cy/Rh ROR1-His k_(a) (M-1Rmax Res % Name s-1) k_(d) (s-1) K_(D) (M) (RU) SD Rmax ATX-P-4531.26E+06 5.91E−03 4.69E−09 529.0 25.6 4.85% (UC961) ATX-P-875 4.93E+053.16E−03 6.45E−09 593.6 22.3 3.76% ATX-P-885 2.70E+05 2.95E−03 1.09E−08341.6 10.2 2.99% ATX-P-890 3.92E+05 3.14E−03 8.40E−09 504.2 15.0 2.97%

TABLE 3 MOUSE ROR-1 BINDING Mouse ROR1-His k_(a) (M-1 Rmax Res % Names-1) k_(d) (s-1) K_(D) (M) (RU) SD Rmax ATX-P- N/A N/A N/A N/A N/A N/A453 (UC961) ATX-P- N/A N/A N/A N/A N/A N/A 875 ATX-P- 2.92E+04 1.32E−034.53E−08 73.5 8.9 12.14% 885 ATX-P- N/A N/A N/A N/A N/A N/A 890

TABLE 4 RAT ROR-1 BINDING Rat ROR1-His k_(a) (M-1 k_(a) (M-1 k_(d) K_(D)Rmax Res % s-1) s-1) (s-1) (M) (RU) SD Rmax N/A N/A N/A N/A N/A N/A N/AN/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/AN/A N/A N/A

TABLE 5 HUMAN ROR-2 BINDING Human ROR2/NTRKR2-His k_(a) (M-1 k_(d) K_(D)Rmax Res % Name s-1) (s-1) (M) (RU) SD Rmax ATX-P- N/A N/A N/A N/A N/AN/A 453 (UC961) ATX-P- N/A N/A N/A N/A N/A N/A 875 ATX-P- N/A N/A N/AN/A N/A N/A 885 ATX-P- N/A N/A N/A N/A N/A N/A 890

TABLE 6 CELLULAR BINDING SUMMARY Cellular Binding Summary Name JeKo-1(Avg EC50) MDA-MB-468 (Avg EC50) ATX-P-453 (UC961) 0.073 0.176 ATX-P-8750.145 0.708 ATX-P-885 1.075 2.508 ATX-P-890 0.174 1.016

TABLE 7 CELLULAR BINDING SUMMARY Cellular Internalization Summary NameJeKo-1 (Avg EC50) MDA-MB-468 (Avg EC50) ATX-P-453 (UC961) 0.073 0.176ATX-P-875 0.145 0.708 ATX-P-885 1.075 2.508 ATX-P-890 0.174 1.016

Example 34

The synthesis of the immunoconjugates was accomplished as set forth inthis example. The antibodies were produced as described in Example 28and were suspended in PBS pH 7.2 with protein concentrations as follows:mAb A at 12.44 mg/ml, mAb B at 13.29 mg/ml, mAb C at 14.90 mg/ml. Forthe reduction and conjugation calculations, a molecular weight of 150000Da for all antibodies was used.

Each antibody was prepared for reduction by the addition of 5% v/v of500 mM Tris, 25 mM EDTA, pH 8.5, followed by the addition of TCEP (6equivlant, 10 mM stock of TCEP in water) and the mixture was maintainedat 20° C. for 2 h.

After DMA was added and gently mixed with the above reduced proteinsolution to achieve a final 10% v/v during conjugation, a toxin-linkerstock solution (12 equivalent, 50 mM in DMA) was added and gently mixed.The bioconjugation was allowed to proceed for approximately 16-20 hovernight at 20° C.; it was complete within 2 h with the extended timeallowed for maleimide ring opening.

The crude conjugate was buffer exchanged to PBS pH 7.4 using a gravityfed NAP 25 (small scale) or a flow HiPrep G25 (large scale) with thecolumns prepared and operated according to manufacturer's (Cytivia)instructions. To remove residual toxin, a 100 mg/ml slurry of activatedcarbon (Sigma/C9157) in PBS pH 7.4 was prepared and added to achieve 1mg carbon to 1 mg starting antibody mass. It was mixed gently for 2 h,sufficiently to maintain the carbon in suspension. Then, the carbon wasremoved by centrifugation at 4000 g. Polysorbate 20 (PS20) was addedfrom a 10% w/v stock solution in PBS pH7.4 to achieve a final 0.02% PS20w/v in the product. The ADC was terminally filtered through a suitablysized 0.2 μm PES filter (chromatography direct/FIL-S-PES-022-13-100-S)under grade A laminar flow. The final product was analysed as follows:monomer and [ADC] mg/ml by SEC HPLC, average DAR by PLRP, residual toxinby RP-HPLC, and endotoxin by Endosafe kinetic chromogenic.

Analytical processes were carried out on HPLC instruments Agilent 1100or 1260 using the protocol set forth below and in Tables 8 and 9.

SEC-HPLC—Monomer Contents and ADC Concentration (Mg/mL)

Column: TOSOH TSKgel G3000SWXL 7.8 mm×30 cm 5 μm particle (MERCK808541)combined with a security guard column (MERCK 822858) with a GFC3000 4×3mm cartridge (Phenomenex)

Buffer: 0.2M Phosphate 0.25M KCl 10% IPA pH 6.95±0.1

Gradient: Isocratic @0.5 ml/min at 25° C.Sample load was approximately 10 μg with monomer and concentrationdetermined from 214 nm signal. Monomer reported based on peakintegration and [ADC] mg/mL based on a calibration curve of antibody.

PLRP-HPLC—DAR Determination

Column: PLRP-S 2.1 mm×5 cm, 5 μm (Agilent PL912-1502)mobile phase A: 0.1% v/v TFA in watermobile phase B: 0.1% v/v TFA in ACN.Gradient: 50° C. at 1 ml/minsample load was 2 μg and analyzed at 214 nm.

TABLE 8 Time % B 0.00 30 2.00 30 10.00 41 11.50 90 15.50 90 16.50 3020.00 30

Residual Toxin Column Kinetex® 2.6 μm C8 100 Å, LC Column 50×4.6 mm,(Phenomex OOB-4497-E0)

Mobile Phase A 0.05% TFA in water

Mobile Phase B 0.05% TFA in CAN

Gradient: 60° C. at 2 ml/min

TABLE 9 Time % B 0.00 5 8.00 95 8.10 100 9.00 100 9.10 5 10.00 5

Analytical sample preparation: A sample (50 μl, ADC or PBS/PS20 matrix)was diluted with 2 μl 5M NaCl, 150 μl cold MeOH (from −20° C. freezer),incubated at −20° C. for 30 min, and centrifuged at 21,000 g at 4° C.for 30 min. Then, the supernatant (125μl) was extracted and mixed with125 μl WFI, 100 μl of this was injected onto the Kinetex column. Datawas analyzed at 214 nm and the residual toxin in the sample estimatedfrom an external calibration curve of the relevant toxin linker. Theresult is expressed as the percentage free relative to free and boundusing the ADC concentration and calculated DAR to determine the amountof bound toxin.

Example 35

Novel ROR-1 antibody-drug conjugates (ADCs) were evaluated by CTG Assayin a similar manner as was described in Example 24 for screening ofpayloads. A total of 3 unique antibodies (mAb A=875, mAb B=885, mAbC=890) were conjugated to 6 separate novel linker/payloads (18-112,19-113, 20-114, 21-120, 22-121 and 23-122) (FIG. 32 ). Briefly ROR+(JeKo-1/MDA-MB-468) or ROR− (Ramos) cells were transferred to 96 wellsand treated with the ADCs in three-fold serial dilution starting from 1mM to 0.0000508 mM (10 points dilution), for 72 h. Cell viability wasanalyzed with CellTiter-Glo® Luminescent Cell Viability Assay (Promega)following the manufactures' instructions (FIG. 33 ). The percentage ofviable cells at each ADC concentration was determined by normalizingwith the luminescence of vehicle control and plotted into percentage ofviability versus dose response curve by nonlinear fit in GraphPad Prismsoftware. The IC50 for each ADC was calculated as the concentration ofcompound killing 50% of cells. Representative assay results aresummarized in TABLE 10. Approximately a 4-fold increase in potency wasachieved with ADCs containing 22-121, 21-120 and 23-122 compared toVLS-101, in ROR+ cells lines. Also in vitro data evidence that mAb A(ATX-P-875) is slightly more potent than the other 2 ROR-1 targetedantibodies B and C (ATX-P-885 and ATX-P-895 respectively).

TABLE 10 Jeko-1 Jeko-1 P3 P4 Avg. ADC #/ Toxin- (RUN1) (RUN2) IC50 ToxinmAb linker IC50 nM IC50 nM nM 1 A 18-122 87.1 113.2 100.1 2 A 21-12014.8 19.0 16.9 3 B 18-122 128.0 183.9 156.0 4 B 21-120 23.4 37.3 30.3 5C 18-112 146.1 197.3 171.7 6 C 21-120 22.6 29.4 26.0 7 UC- 18-112 181.2218.2 199.7 961 8 UC- 21-120 28.3 40.5 34.4 961 9 A 19-113 146.3 164.4155.4 10 A 22-121 11.2 12.7 11.9 11 B 19-113 172.1 197.2 184.7 12 B22-121 17.1 18.3 17.7 13 C 19-113 177.0 212.4 194.7 14 C 22-121 19.719.1 19.4 15 A 20-114 115.4 120.8 118.1 16 A 23-122 13.6 16.3 14.9 17 B20-114 112.2 106.2 109.2 18 B 23-122 23.4 24.9 24.1 19 C 20-114 119.1126.5 122.8 20 C 23-122 22.2 29.8 26.0 21 UC- 23-122 25.1 31.0 28.1 691VLS- mc-vc- MMAE 63.4 56.8 60.1 101 PAB MMAE 0.2 0.2 0.2 Dxd 0.9 0.95-48 0.5 0.5 0.5 9-74 2.4 3.7 3.1

Furthermore, although the foregoing has been described in some detail byway of illustrations and examples for purposes of clarity andunderstanding, it will be understood by those of skill in the art thatnumerous and various modifications can be made without departing fromthe spirit of the present disclosure. Therefore, it should be clearlyunderstood that the forms disclosed herein are illustrative only and arenot intended to limit the scope of the present disclosure, but rather toalso cover all modification and alternatives coming with the true scopeand spirit of the disclosure.

SEQUENCE LISTING ATX-P-875 VH CDR1 (Kabat) SEQ ID NO: 1 GFTFSNAWATX-P-875 VH CDR2 (Kabat) SEQ ID NO: 2 IKSKTDGGTTATX-P-875 VH CDR3 (Kabat) SEQ ID NO: 3 TTGPDDLDY ATX-P-875 VH ntSEQ ID NO: 4 GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATTAAAAGCAAAACTGATGGTGGGACAACAGACTACGCTGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCAAAAAACACGCTCTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTACCACAGGCCCTGACGATCTTGACTACTGGGGCCAGGGAACCCCGGTCACCGTCTCCTCA ATX-P-875 VH AA SEQ ID NO: 5AAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTGP DDLDYWGQGTPVTVSSATX-P-875 HC IgG1-Fc nt SEQ ID NO: 6GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATTAAAAGCAAAACTGATGGTGGGACAACAGACTACGCTGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCAAAAAACACGCTCTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTACCACAGGCCCTGACGATCTTGACTACTGGGGCCAGGGAACCCCGGTCACCGTCTCCTCAGCTAGCACTAAAGGGCCTTCTGTATTTCCCTTGGCCCCGTCCAGCAAATCGACCTCGGGAGGGACAGCCGCCCTGGGTTGCCTTGTGAAAGATTATTTCCCTGAGCCAGTTACCGTAAGTTGGAACAGTGGGGCGCTGACAAGTGGTGTGCACACGTTTCCTGCCGTCCTGCAATCATCGGGCTTGTATAGCCTCAGCTCTGTGGTCACTGTCCCAAGTTCATCGCTGGGCACTCAGACGTATATTTGCAATGTGAACCACAAACCTTCAAATACAAAAGTGGATAAACGCGTAGAACCGAAATCGTGTGATAAAACTCACACATGCCCGCCATGCCCGGCACCTGAACTGCTTGGTGGTCCCAGCGTGTTCCTGTTCCCGCCGAAGCCTAAAGATACTCTAATGATCAGCCGTACGCCAGAGGTGACATGTGTCGTGGTTGACGTGTCCCACGAAGATCCCGAAGTTAAGTTCAATTGGTATGTTGATGGTGTAGAGGTACACAATGCTAAGACTAAACCTCGCGAGGAGCAGTACAATTCGACCTATCGTGTCGTGAGCGTTCTGACCGTCCTTCACCAAGATTGGCTTAACGGCAAAGAATATAAGTGCAAGGTAAGCAATAAAGCACTTCCGGCTTCCGCCTTCTCGTGAGGAAATGACTAAAAATCAAGTATCCCTTACGTGTCTGGTTAAAGGTTTTTATCCTAGCGATATTGCTGTTGAATGGGAATCGAACGGTCAGCCGGAGAATAATTATAAAACAACGCCACCCGTCCTGGATAGCGACGGCTCATTTTTTCTGTATAGCAAACTGACTGTAGATAAATCACGGTGGCAGCAGGGCAATGTATTCAGTTGCTCCGTTATGCATGAAGCGTTACATAATCACTACACGCAGAAATCTCTTAGTCTTTCACCCGGT ATX-P-875 HC IgG1-Fc AASEQ ID NO: 7 AAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTGPDDLDYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGATX-P-875 VL CDR1 (Kabat) SEQ ID NO: 8 QSISSY ATX-P-875 VL CDR2 (Kabat)AAS ATX-P-875 VL CDR3 (Kabat) SEQ ID NO: 10 QQYDNLPIT ATX-P-875 VL ntSEQ ID NO: 11 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTATGATAATCTCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAA A ATX-P-875 VL AA SEQ ID NO: 12DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQYDNLPITFGQGTRLEIKATX-P-875 Kappa LC nt SEQ ID NO: 13GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTATGATAATCTCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGTACGGTAGCTGCCCCTTCAGTTTTTATCTTTCCGCCGTCTGACGAGCAGTTAAAATCCGGGACCGCTTCTGTAGTTTGCCTGCTGAATAATTTTTATCCGCGTGAGGCTAAAGTACAATGGAAAGTCGACAATGCTTTGCAGTCGGGAAATTCACAGGAAAGTGTTACGGAGCAGGATTCTAAAGATTCCACATATTCACTCAGCTCCACCCTTACACTGAGCAAAGCCGACTATGAAAAACATAAAGTTTACGCATGTGAGGTGACGCACCAAGGATTATCCAGTCCGGTCACAAAATCGTTTAACCGCGGTGAGT GT ATX-P-875 Kappa LC AASEQ ID NO: 14 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDNLPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECATX-P-885 VH CDR1 (Kabat) SEQ ID NO: 15 GGSFSGYYATX-P-885 VH CDR2 (Kabat) SEQ ID NO: 16 INHSGSTATX-P-885 VH CDR3 (Kabat) SEQ ID NO: 17 AREGVYEDY ATX-P-885 VH ntSEQ ID NO: 18 CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTATATTACTGTGCGAGAGAGGGTGTCTACGAGGACTACTGGGGCCA GGGAACCCTGGTCACCGTCTCCTCAATX-P-885 VH AA SEQ ID NO: 19 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGVYEDYWGQGTLVTVSS ATX-P-885 HC IgG1-Fc ntSEQ ID NO: 20 CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTATATTACTGTGCGAGAGAGGGTGTCTACGAGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACTAAAGGGCCTTCTGTATTTCCCTTGGCCCCGTCCAGCAAATCGACCTCGGGAGGGACAGCCGCCCTGGGTTGCCTTGTGAAAGATTATTTCCCTGAGCCAGTTACCGTAAGTTGGAACAGTGGGGCGCTGACAAGTGGTGTGCACACGTTTCCTGCCGTCCTGCAATCATCGGGCTTGTATAGCCTCAGCTCTGTGGTCACTGTCCCAAGTTCATCGCTGGGCACTCAGACGTATATTTGCAATGTGAACCACAAACCTTCAAATACAAAAGTGGATAAACGCGTAGAACCGAAATCGTGTGATAAAACTCACACATGCCCGCCATGCCCGGCACCTGAACTGCTTGGTGGTCCCAGCGTGTTCCTGTTCCCGCCGAAGCCTAAAGATACTCTAATGATCAGCCGTACGCCAGAGGTGACATGTGTCGTGGTTGACGTGTCCCACGAAGATCCCGAAGTTAAGTTCAATTGGTATGTTGATGGTGTAGAGGTACACAATGCTAAGACTAAACCTCGCGAGGAGCAGTACAATTCGACCTATCGTGTCGTGAGCGTTCTGACCGTCCTTCACCAAGATTGGCTTAACGGCAAAGAATATAAGTGCAAGGTAAGCAATAAAGCACTTCCGGCCCCAATCGACTCGTGAGGAAATGACTAAAAATCAAGTATCCCTTACGTGTCTGGTTAAAGGTTTTTATCCTAGCGATATTGCTGTTGAATGGGAATCGAACGGTCAGCCGGAGAATAATTATAAAACAACGCCACCC GTCCTGGATAGCGACGGCTCATTTTTTCTATX-P-885 HC IgG1-Fc AA SEQ ID NO: 21QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGVYEDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG ATX-P-885 VL CDR1 (Kabat)SEQ ID NO: 22 QSVSNY ATX-P-885 VL CDR2 (Kabat) DAYATX-P-885 VL CDR3 (Kabat) SEQ ID NO: 24 QQRSNWPLT ATX-P-885 VL ntSEQ ID NO: 25 GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAACTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCCTACAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACCTTCGGCCAAGGGACACGACTGGAGATTAA A ATX-P-885 VL AA SEQ ID NO: 26EIVLTQSPATLSLSPGERATLSCRASQSVSNYLAWYQQKPGQAPRLLIYDAYNRATGIPARFSGSGSGTDFTLTISSLEP EDFAVYYCQQRSNWPLTFGQGTRLEIKATX-P-885 Kappa LC nt SEQ ID NO: 27GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAACTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCCTACAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGTACGGTAGCTGCCCCTTCAGTTTTTATCTTTCCGCCGTCTGACGAGCAGTTAAAATCCGGGACCGCTTCTGTAGTTTGCCTGCTGAATAATTTTTATCCGCGTGAGGCTAAAGTACAATGGAAAGTCGACAATGCTTTGCAGTCGGGAAATTCACAGGAAAGTGTTACGGAGCAGGATTCTAAAGATTCCACATATTCACTCAGCTCCACCCTTACACTGAGCAAAGCCGACTATGAAAAACATAAAGTTTACGCATGTGAGGIGACGCACCAAGGATTATCCAGTCCGGTCACAAAATCGTTTAACCGCGGTGAGT GT ATX-P-885 Kappa LC AASEQ ID NO: 28 EIVLTQSPATLSLSPGERATLSCRASQSVSNYLAWYQQKPGQAPRLLIYDAYNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECATX-P-890 VH CDR1 (Kabat) SEQ ID NO: 29 GYTFTGYYATX-P-890 VH CDR2 (Kabat) SEQ ID NO: 30 INPNSGGTATX-P-890 VH CDR3 (Kabat) SEQ ID NO: 31 VRDQVQLERFDS ATX-P-890 VH ntSEQ ID NO: 32 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGTGAGAGATCAGGTACAACTGGAACGGTTCGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA ATX-P-890 VH AA SEQ ID NO: 33QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCVRDQVQLERFDSWGQGTLVTVSS ATX-P-890 HC IgG1-Fc ntSEQ ID NO: 34 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGTGAGAGATCAGGTACAACTGGAACGGTTCGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACTAAAGGGCCTTCTGTATTTCCCTTGGCCCCGTCCAGCAAATCGACCTCGGGAGGGACAGCCGCCCTGGGTTGCCTTGTGAAAGATTATTTCCCTGAGCCAGTTACCGTAAGTTGGAACAGTGGGGCGCTGACAAGTGGTGTGCACACGTTTCCTGCCGTCCTGCAATCATCGGGCTTGTATAGCCTCAGCTCTGTGGTCACTGTCCCAAGTTCATCGCTGGGCACTCAGACGTATATTTGCAATGTGAACCACAAACCTTCAAATACAAAAGTGGATAAACGCGTAGAACCGAAATCGTGTGATAAAACTCACACATGCCCGCCATGCCCGGCACCTGAACTGCTTGGTGGTCCCAGCGTGTTCCTGTTCCCGCCGAAGCCTAAAGATACTCTAATGATCAGCCGTACGCCAGAGGTGACATGTGTCGTGGTTGACGTGTCCCACGAAGATCCCGAAGTTAAGTTCAATTGGTATGTTGATGGTGTAGAGGTACACAATGCTAAGACTAAACCTCGCGAGGAGCAGTACAATTCGACCTATCGTGTCGTGAGCGTTCTGACCGTCCTTCACCAAGATTGGCTTAACGGCAAAGAATATAAGTGCAAGGTAAGCAATAAAGCACTTCCGGCCCCAATCGAGAAAACCATTTCCAAGGCCAAAGGTCAACCAAGAGAACCCCAGGTGTATACTCTTCCGCCTTCTCGTGAGGAAATGACTAAAAATCAAGTATCCCTTACGTGTCTGGTTAAAGGTTTTTATCCTAGCGATATTGCTGTTGAATGGGAATCGAACGGTCAGCCGGAGAATAATTATAAAACAACGCCACCCGTCCTGGATAGCGACGGCTCATTTTTTCTGTATAGCAAACTGACTGTAGATAAATCACGGTGGCAGCAGGGCAATGTATTCAGTTGCTCCGTTATGCATGAAGCGTTACATAATCACTACACGCAG AAATCTCTTAGTCTTTCACCCGGTATX-P-890 HC IgG1-Fc AA SEQ ID NO: 35QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCVRDQVQLERFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGATX-P-890 VL CDR1 (Kabat) SEQ ID NO: 36 QDISNY ATX-P-890 VL CDR2 (Kabat)DAS ATX-P-890 VL CDR3 (Kabat) SEQ ID NO: 38 QQYDNLPPT ATX-P-890 VL ntSEQ ID NO: 39 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAATCTCCCTCCCACTTTCGGCCCTGGGACCAAGGTGGAAATCAA A ATX-P-890 VL AA SEQ ID NO: 40DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQP EDIATYYCQQYDNLPPTFGPGTKVEIKATX-P-890 Kappa LC nt SEQ ID NO: 41GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAATCTCCCTCCCACTTTCGGCCCTGGGACCAAGGTGGAAATCAAACGTACGGTAGCTGCCCCTTCAGTTTTTATCTTTCCGCCGTCTGACGAGCAGTTAAAATCCGGGACCGCTTCTGTAGTTTGCCTGCTGAATAATTTTTATCCGCGTGAGGCTAAAGTACAATGGAAAGTCGACAATGCTTTGCAGTCGGGAAATTCACAGGAAAGTGTTACGGAGCAGGATTCTAAAGATTCCACATATTCACTCAGCTCCACCCTTACACTGAGCAAAGCCGACTATGAAAAACATAAAGTTTACGCATGTGAGGTGACGCACCAAGGATTATCCAGTCCGGTCACAAAATCGTTTAACCGCGGTGAGT GT ATX-P-890 Kappa LC AASEQ ID NO: 42 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPPTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECTetrapeptide residue 1 SEQ ID NO: 43 GGFG Tetrapeptide residue 2SEQ ID NO: 44 EGGF Tetrapeptide residue 3 SEQ ID NO: 45 SGGFTetrapeptide residue 4 SEQ ID NO: 46 KGGF

1. An immunoconjugate having Formula (I),Ab-[S-L¹-L²-L³-L⁴-L⁵-L⁶-L⁷-D]_(n)   (I) wherein: Ab is an antibody or anantigen-binding fragment thereof;

L¹ is L² is absent,

Z¹ and Z² are each individually hydrogen, halogen, NO₂, —O—(C₁-C₆alkyl), or C₁-C₆alkyl; L³ is —(CH₂)n¹-C(═O)— or—(CH₂CH₂O)n¹-(CH₂)n¹C(═O)—; each n¹ is independently an integer of 0 to12; L⁴ is a tetrapeptide residue; L⁵ is absent or —[NH(CH₂)n²]_(n) ³—;n² is an integer of 0 to 6; n³ is an integer of 0 to 2; L⁶ is absent or

L⁷ is absent,

D is a drug moiety; and n is an integer from 1 to 10, wherein saidantibody or binding fragment thereof is selected from: an antibody orbinding fragment thereof comprising: a heavy chain comprising: VHCDR 1comprising an amino acid sequence of SEQ ID NO:29; VHCDR 2 comprising anamino acid sequence of SEQ ID NO:30; and VHCDR 3 comprising an aminoacid sequence of SEQ ID NO:31; and a light chain comprising: VLCDR 1comprising an amino acid sequence of SEQ ID NO:36; VLCDR 2 comprising anamino acid sequence of DAS; and VLCDR 3 comprising an amino acidsequence of SEQ ID NO:38; wherein the antibody or antigen-bindingfragment specifically binds to the extracellular domain of humanreceptor tyrosine kinase like orphan receptor 1 (ROR1). 2.-20.(canceled)
 21. The immunoconjugate of claim 1, wherein D is a drugmoiety of Formula (II) having the structure:

wherein: R¹ and R² are each individually selected from the groupconsisting of hydrogen, halogen, —CN, —OR⁵, —NR⁵R⁶, a substituted or anunsubstituted C₁-C₆ alkyl, a substituted or an unsubstituted C₁-C₆haloalkyl, a substituted or an unsubstituted —O—(C₁-C₆ alkyl), asubstituted or an unsubstituted —O—(C₁-C₆ haloalkyl), and—[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃, or a substituted or an unsubstituted—O—(CR⁵R⁶)_(m)—O— such that R¹ and R² taken together form a ring; R³ ishydrogen or a substituted or an unsubstituted C₁-C₆ alkyl, a substitutedor an unsubstituted C₁-C₆ haloalkyl, or —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃;R⁴ is hydrogen, a substituted or an unsubstituted —(C₁-C₆ alkyl)-X², asubstituted or an unsubstituted —(C₁-C₆ haloalkyl)-X², a substituted oran unsubstituted —(C₁-C₆ alkenyl)-X², a substituted or an unsubstituted—(C₁-C₆ haloalkenyl)-X², a substituted or an unsubstituted —(C₁-C₆alkynyl)-X², or a substituted or an unsubstituted —(C₁-C₆haloalkynyl)-X²; X¹ is —O—, —S(O_(n6))—, —NH—, —O—(C═O)—, —NH—(C═O)—,—NH—(C═O)—O—, —NH—(C═O)—NH—, or —NH—S(O_(n6))—; X² is —OR⁹, —SR⁹, or—NHR⁹; R⁵ and R⁶ are each individually hydrogen, halogen, a substitutedor an unsubstituted C₁-C₆ alkyl, a substituted or an unsubstituted C₁-C₆haloalkyl, or —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₃; m is 1 or 2; n⁴ and n⁵ areeach individually 0, 1 or 2, with the proviso that n⁴ and n⁵ are notboth 0; n⁶ is 0, 1 or 2; each Y is individually H or halogen; each p isindividually 1, 2, 3, 4, 5, or 6; each q is individually 0, 1, 2, 3, 4,5, or 6; each t is individually 1, 2, 3, 4, 5, or 6; R⁷ is H, —COR⁸,—CO₂R⁸, —(CO)—NHR⁸, L⁴, L⁵, L⁶, or L⁷; R⁸ is a substituted or anunsubstituted C₁-C₆ alkyl-X³, a substituted or an unsubstituted C₁-C₆haloalkyl-X³, or —[(CY₂)_(p)O(CY₂)_(q)]_(t)CY₂—X³; R⁹ is H, —COR⁸,—CO₂R⁸, —(CO)—NHR⁸, L⁴, L⁵, L⁶, or L⁷, with the proviso that exactly oneof R⁷ and R⁹ is L⁴, L⁵, L⁶, or L⁷; and each X³ is individually —H, —OH,—SH, or —NH₂.
 22. (canceled)
 23. (canceled)
 24. (canceled) 25.(canceled)
 26. A compound of Formula (II), or a pharmaceuticallyacceptable salt thereof, having the structure:

wherein: R¹ and R² are each individually selected from the groupconsisting of hydrogen, halogen, —OR⁵, an unsubstituted C₁-C₆ alkyl, anunsubstituted C₁-C₆ haloalkyl, and an unsubstituted —O—(C₁-C₆ alkyl), oran unsubstituted —O—(CR⁵R⁶)_(m)—O— such that R¹ and R² taken togetherform a ring; R³ is hydrogen or an unsubstituted C₁-C₆ alkyl; R⁴ ishydrogen or an unsubstituted —(C₁-C₆ alkyl)-X²; X¹ is —O—; X² is —OH; R⁵and R⁶ are each individually hydrogen; R⁷ is H; m is 1; n⁴ is 1 or 2;and n⁵ is 0; with the proviso that Formula (II) does not representexatecan.
 27. The compound of claim 26, or a pharmaceutically acceptablesalt thereof, wherein at least one of R¹ and R² is hydrogen.
 28. Thecompound of claim 26, or a pharmaceutically acceptable salt thereof,wherein at least one of R¹ and R² is halogen.
 29. The compound of claim26, or a pharmaceutically acceptable salt thereof, wherein at least oneof R¹ and R² is an unsubstituted C₁-C₆ alkyl.
 30. (canceled) 31.(canceled)
 32. (canceled)
 33. The compound of claim 26, or apharmaceutically acceptable salt thereof, wherein R¹ and R² are eachindividually selected from the group consisting of hydrogen, fluoro,methoxy, methyl, and difluoromethyl, or —O—(CH₂)—O— such that R¹ and R²taken together form a ring.
 34. The compound of claim 26, or apharmaceutically acceptable salt thereof, wherein R³ is hydrogen. 35.The compound of claim 26, or a pharmaceutically acceptable salt thereof,wherein R³ is an unsubstituted C₁-C₆ alkyl.
 36. The compound of claim35, or a pharmaceutically acceptable salt thereof, wherein R³ is methyl.37. The compound of claim 26, or a pharmaceutically acceptable saltthereof, wherein R⁴ is hydrogen.
 38. The compound of claim 26, or apharmaceutically acceptable salt thereof, wherein R⁴ is an unsubstituted—(C₁-C₆ alkyl)-X². 39.-44. (canceled)
 45. A compound, or apharmaceutically acceptable salt thereof, represented by Formula (IVa):

wherein: R¹ and R² are each individually selected from the groupconsisting of hydrogen, halogen, —OR⁵, an unsubstituted C₁-C₆ alkyl, anunsubstituted C₁-C₆ haloalkyl and an unsubstituted —O—(C₁-C₆ alkyl), oran unsubstituted —O—(CR⁵R⁶)_(m)—O— such that R¹ and R² taken togetherform a ring; R³ is hydrogen or an unsubstituted C₁-C₆ alkyl; R⁴ ishydrogen or an unsubstituted —(C₁-C₆ alkyl)-X²; X¹ is —O—; X² is —OH; R⁵and R⁶ are each individually hydrogen; R⁷ is H; and m is
 1. 46.(canceled)
 47. (canceled)
 48. The compound of claim 26, or apharmaceutically acceptable salt thereof, wherein the compound ofFormula (JI) is selected from the group consisting of:


49. (canceled)
 50. A method for treating a cancer or a tumor comprisingadministering an effective amount of a compound of claim 26, or apharmaceutically active salt thereof, to a subject having the cancer orthe tumor.
 51. The method of claim 50, wherein the cancer or the tumoris selected from lung cancer, urothelial cancer, colorectal cancer,prostate cancer, ovarian cancer, pancreatic cancer, breast cancer,bladder cancer, gastric cancer, gastrointestinal stromal tumor, uterinecervix cancer, esophageal cancer, squamous cell carcinoma, peritonealcancer, liver cancer, hepatocellular cancer, colon cancer, rectalcancer, colorectal cancer, endometrial cancer, uterine cancer, salivarygland cancer, kidney cancer, vulval cancer, thyroid cancer, peniscancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma.52. (canceled)
 53. (canceled)
 54. A conjugate having Formula (III),Mi-L²-L³-L⁴-L⁵-L⁶-L⁷-D   (III) wherein: Mi is

L² is absent,

Z¹ and Z² are each individually hydrogen, halogen, NO₂, —O—(C₁-C₆alkyl), or C₁-C₆alkyl; L³ is —(CH₂)n¹-C(═O)—; n¹ is an integer of 0 to12; L⁴ is a tetrapeptide residue; L⁵ is absent or —[NH(CH₂)n²]_(n) ³—;n² is an integer of 0 to 6; n³ is an integer of 0 to 2; L⁶ is absent; L⁷is absent,

D is a drug moiety, wherein D is a compound of Formula (II) having thestructure: wherein:

R¹ and R² are each individually selected from the group consisting ofhydrogen, halogen, —OR⁵, an unsubstituted C₁-C₆ alkyl, an unsubstitutedC₁-C₆ haloalkyl and an unsubstituted —O—(C₁-C₆ alkyl), or anunsubstituted —O—(CR⁵R⁶)_(m)—O— such that R¹ and R² taken together forma ring; R³ is hydrogen or an unsubstituted C₁-C₆ alkyl; R⁴ is hydrogenor an unsubstituted —(C₁-C₆ alkyl)-X²; X¹ is —O—; X² is —OR⁹; R⁵ and R⁶are each individually hydrogen; m is 1; n⁴ is 1 or 2; n⁵ is 0; R⁷ is H;and R⁹ is L⁴, L⁵, L⁶, or L⁷. 55.-73. (canceled)
 74. The conjugate ofclaim 54, wherein D is a compound of Formula (II) having the structure:

wherein: R¹ is methyl; R² is fluoro; R³ is hydrogen; R⁴ is anunsubstituted —(C₂alkyl)-X²; and n⁴ is
 2. 75.-113. (canceled)
 114. Theimmunoconjugate of claim 21, wherein D is a drug moiety of Formula (II)having the structure:

wherein: R¹ and R² are each individually selected from the groupconsisting of hydrogen, halogen, —OR⁵, an unsubstituted C₁-C₆ alkyl, anunsubstituted C₁-C₆ haloalkyl and an unsubstituted —O—(C₁-C₆ alkyl), oran unsubstituted —O—(CR⁵R⁶)_(m)—O— such that R¹ and R² taken togetherform a ring; R³ is hydrogen or an unsubstituted C₁-C₆ alkyl; R⁴ ishydrogen or an unsubstituted —(C₁-C₆ alkyl)-X²; X¹ is —O—; X² is —OR⁹;R⁵ and R⁶ are each individually hydrogen; m is 1; n⁴ is 1 or 2; n⁵ is 0;R⁷ is H; and R⁹ is L⁴, L⁵, L⁶, or L⁷.
 115. The immunoconjugate of claim114, wherein n⁴ is
 1. 116. The immunoconjugate of claim 114, wherein n⁴is
 2. 117. The immunoconjugate of claim 1, wherein the immunoconjugateis:

wherein Z¹ and Z² are each individually hydrogen.
 118. The compound ofclaim 26, wherein n⁴ is
 1. 119. The compound of claim 26, wherein n⁴ is2.
 120. The compound of claim 74, wherein n⁴ is
 1. 121. The compound ofclaim 74, wherein n⁴ is
 2. 122. The compound of claim 26, or apharmaceutically acceptable salt thereof, wherein the compound ofFormula (II) is


123. The conjugate of claim 54, wherein: L² is

Z¹ and Z² are each individually hydrogen; n¹ is the integer 2; L⁴ isgly-gly-phe-gly; L⁵ is —[NH(CH₂)n²]n³-; n² is the integer of 1; n³ isthe integer of 1; and L⁷ is absent.
 124. The conjugate of claim 54,wherein Formula (III) is:


125. The compound of claim 45, or pharmaceutically acceptable saltthereof, wherein the compound of Formula (IVa) is:

 or a pharmaceutically acceptable salt thereof.
 126. The conjugate ofclaim 54, wherein Formula (III) is:


127. The conjugate of claim 54, wherein Formula (III) is:


128. The conjugate of claim 54, wherein D is a compound of Formula (II)having the structure: wherein:

R¹ is methyl; R² is fluoro; R³ is hydrogen; R⁴ is an unsubstituted —(C₂alkyl)-X²; and n⁴ is
 2. 129. The conjugate of claim 123, wherein D is acompound of Formula (II) having the structure: wherein:

R¹ is methyl; R² is fluoro; R³ is hydrogen; R⁴ is an unsubstituted —(C₂alkyl)-X²; and n⁴ is
 2. 130. The conjugate of claim 54, wherein: L² isabsent; n¹ is the integer 5; L⁴ is gly-gly-phe-gly; L⁵ is—[NH(CH₂)n²n³-; n² is the integer of 1; n³ is the integer of 1; and L⁷is absent.
 131. The conjugate of claim 130, wherein D is a compound ofFormula (II) having the structure:

wherein: R¹ is methyl; R² is fluoro; R³ is hydrogen; R⁴ is anunsubstituted —(C₂ alkyl)-X²; and n⁴ is
 2. 132. The conjugate of claim54, wherein Formula (III) is:


133. The immunoconjugate of claim 1, wherein the immunoconjugate is:


134. The immunoconjugate of claim 1, wherein the immunoconjugate is:


135. The immunoconjugate of claim 21, wherein D is a compound of Formula(II) having the structure:


136. The immunoconjugate of claim 135, wherein D is a compound ofFormula (II) having the structure:


137. The immunoconjugate of claim 135, wherein D is a compound ofFormula (II) having the structure:


138. The compound of claim 122, or a pharmaceutically acceptable saltthereof, wherein the compound of Formula (II) is


139. The compound of claim 122, or a pharmaceutically acceptable saltthereof, wherein the compound of Formula (II) is